Linköping University Medical Dissertations No. 1493

APPLICATIONS OF HUMAN SKIN IN VITRO

Susanna Lönnqvist

Division of Clinical Sciences Department of Clinical and Experimental Medicine

Medical Faculty Linköping, 2016

© Susanna Lönnqvist, 2016

ISBN: 978-91-7685-895-0

ISSN: 0345-0082

Papers III and IV are reprinted with the permission of Taylor & Francis

During the course of the research underlying this thesis, the author was enrolled in

Forum Scientium, a multidisciplinary doctoral programme at Linköping

University, Sweden.

Printed by LiU-Tryck, Linköping, Sweden December 2015

Siis älä siitä huoli, näinhän täällä käy

Aurinkokin paistaa, vaikkei sitä näy

(Eppu Normaali, 1993)

So don’t you worry about it, that’s the way things go

The sun is still shining, even though it doesn’t show

SUPERVISOR

Gunnar Kratz, Professor

Department of Clinical and Experimental Medicine

Linköping University

CO-SUPERVISOR

Magnus Berggren, Professor

Department of Science and Technology

Linköping University

FACULTY OPPONENT

Jyrki Vuola, Associate Professor

Institute of Clinical Medicine

Helsinki University

ABSTRACT

Chronic wounds are a substantial problem in today’s health care and place

significant strains on the patient. Successful modelling of the wound healing

process is pivotal for the advancement of wound treatment research. Wound

healing is a dynamic and multifactorial process involving all constituents of the

skin. The progression from haemostasis and inflammation to proliferation of

epidermal keratinocytes and dermal fibroblasts, and final scar maturation can be

halted and result in a chronic wound that fails to re-epithelialise. The wound

healing process constitutes an example of dynamic reciprocity in tissue where

cellular changes take place on cues from the extracellular matrix and vice versa

when tissue homeostasis is disturbed. The extracellular matrix provides a structural

context for the resident cells and the epidermal keratinocytes, and a functioning

interplay between the two tissue compartments is crucial for successful wound

healing to take place. Work included in this thesis has applied viable human full

thickness skin in vitro to investigate the re-epithelialisation process and barrier

function of intact skin. The use of full thickness skin in vitro can take into account

the contextual aspect of the process where the epidermal keratinocytes are

activated and obtain a migratory phenotype, and are continuously dependent on

the cues from the extracellular matrix and support of the dermis. When utilising

skin for studies on re-epithelialisation, circular standardised full thickness wounds

were created and cultured for up to four weeks in tissue culture. In paper I, the

organisation of a thick neoepidermis was investigated in the in vitro wound healing

model when resident cells were provided with a porous suspended three

dimensional gelatin scaffold. In paper II we investigated the use of a fluorescent

staining conventionally used for proliferation studies to facilitate the tracing of

transplanted epidermal cells in in vitro wounds, in order to improve and expand

the use of the model. In paper III the model was utilised to investigate the treatment

approach of acidification of wounds to evaluate the suitability of such intervention

in regards to keratinocyte function and re-epithelialisation. Studies on re-

epithelialisation with the aid of the in vitro wound healing model provided insight

in neoepidermal structure with porous gelatin scaffolding in the wound, a novel

methodological approach to tracing cells and response to constrained wound

healing environment. In paper IV, intact human skin was evaluated for modelling

the cytotoxic response after exposure to a known irritant compound. To study

barrier function, intact skin was exposed to irritants by restricting exposure

topically, and full thickness skin in vitro was found suitable for modelling

cytotoxicity responses. Employing human full thickness skin in vitro makes use of

the actual target tissue of interest with epidermal and dermal cells, and full barrier

function.

Populärvetenskaplig sammanfattning

Då huden skadas och ett sår bildas startas en mängd processer för att hejda

ytterligare skada, begränsa infektion och reparera vävnaden för att återskapa den

starka barriär som huden bildar mot omvärlden. Sårläkning är en dynamisk process

som involverar alla celler och komponenter i huden. Läkningen startar med att

blodflödet stillas och ett inflammatoriskt svar tar vid för att rengöra det skadade

området. Cellerna i sårets kanter och i underliggande vävnad aktiveras för att

reparera skadan. Översta hudlagret, epidermis, består av flera lager av epidermala

keratinocyter. Denna celltyp ser till att den strukturella barriären som huden utgör

är intakt genom att det i nedersta lagret av epidermis hela tiden bildas nya

keratinocyter. Dessa genomgår sedan en process som till slut bildar ett flerskikat

lager av keratinocyter med döda och tåliga keratinocyter allra ytterst. Vid

uppkomsten av ett sår aktiveras keratinocyterna och får egenskaper de annars inte

uppvisar. De blir mobila då deras mål är att flytta sig ut från sårkanterna, bli flera

och till slut täcka över såret med keratinocyter och slutligen ny epidermis.

Samtidigt som keratinocyterna täcker in såret jobbar cellerna i det undre hudlagret

dermis med att utsöndra komponenter för att möjliggöra keratinocyternas vandring

ut från sårkanterna. Dessa celler heter dermala fibroblaster, och de fortsätter med

att utsöndra beståndsdelar för den nya vävnaden och bidrar till att bilda de delar

som till slut blir ett ärr i huden. De epidermala keratinocyterna och de dermala

fibroblasterna är beroende av varandra i sårläkningsprocessen, och de signallerar

till varandra och omgivande vävnad under hela förloppet.

Avhandlingens arbeten har riktat in sig på att undersöka hur man kan använda

mänsklig hud i laboratoriet för att undersöka sårläkningsprocessen och

barriärfunktionen hos hud. Genom att använda sig av hud i en

laboratorieuppställning kan man studera händelseförloppet och delar av det genom

att ändra vissa detaljer, införa behandling eller ändra miljön som huden och

cellerna i den hålls i. Användning av mänsklig hud möjliggör iakttagandet av

samarbetet mellan keratinocyterna och fibroblasterna, och de olika delarna av

huden de finns i, vilket inte är möjligt i förenklade modeller av hud. Att hitta bra

och representativa sätt att följa sårläkning är viktigt för att hitta mekanismer vid

bildning av sår som inte läker och behandling av dem.

Populaaritieteellinen tiivistelmä

Kun iho vahingoittuu ja syntyy haava, käynnistyy samanaikaisesti useita eri

prosesseja haavan parantamiseksi: lisävaurioita pitää välttää, infektioriskiä

pienentää ja fyysinen suoja pitää luoda uudestaan. Haavan parantuminen on

dynaaminen prosessi johon osallistuvat kaikki ihon eri solutyypit ja osat.

Parantuminen alkaa verenvuodon pysähtymisellä ja tulehdusvasteella, jotta

vaurioitunut kudos pysyy puhtaana. Ihosolut haavan reunoissa ja alla olevassa

kudoksessa aktivoituvat. Ihon ylin kerros, orvaskesi, koostuu keratinosyyteistä.

Tämä solutyyppi pitää huolen ihon rakenteellisesta suojasta siten, että orvaskeden

alimmassa kerroksessa syntyy jatkuvasti uusia keratinosyyttejä. Nämä solut

käyvät läpi eri kehitysmuotoja, jotka johtavat kerroksittaiseen orvaskeden

rakenteeseen, ja muodostavat lopuksi orvaskeden ylimmän kerrokseen, jonka solut

ovat kuolleita ja hyvin kestäviä. Kun iho vahingoittuu, keratinosyytit aktivoituvat

ja ryhtyvät toimimaan normaalista poikkeavalla tavalla. Keratinosyytit muutuvat

liikkuviksi ja niiden tavoitteena on siirtyä haavan reunasta peittämään haava-

aluetta. Keratinosyyttejä syntyy lisää ja lopulta koko haavan alue on niiden

peitossa ja uuden orvaskeden muodostuminen voi alkaa. Samaan aikaan kun

keratinosyytit vaeltavat haavojen reunoilta poispäin, solut alla olevassa

haavakudoksessa tuottavat kudososia jotka mahdollistavat tämän vaelluksen.

Nämä solut ovat fibroblasteja, sidekudoksessa yleisesti esiintyvää solutyyppiä.

Fibroblastit jatkavat uuden sidekudoksen osien tuottamisen ja avustavat niiden

osien kanssa, jotka lopulta muodostavat arven ihossa. Keratinosyytit ja fibroblastit

ovar riippuvaisia toisistaan koko haavan parantumisen keston ajan ja

kommunikoivat toistensa kanssa prosessin kuluessa.

Tässä väitöskirjassa keskitytään tutkimaan miten ihmisihoa voi käyttää

laboratoriossa jotta haavan parantumista ja ihon suojan muodostamista voitaisiin

tutkia. Ihon käyttö laboratoriossa mahdollistaa haavan parantumisen eri vaiheiden

tutkimisen, koska prosessin eri osia voi silloin muunnella tai niihin voi vaikuttaa

hallitusti. Ihon käyttö mahdollistaa myös keratinosyyttien ja fibroblastien

yhteistyön tutkimisen, mikä ei ole mahdollista yksinkertaisemmissa iho-malleissa.

Hyvien ja edustavien mallintamismuotojen löytyminen on tärkeää kroonisten

haavojen ja niiden hoitomuotojen tutkimisessa.

Table of contents

LIST OF PAPERS ................................................................................................. 1

Abbreviations ..................................................................................................... 3

INTRODUCTION ................................................................................................... 5

The structure of the epidermis is linked to the life cycle of the keratinocyte ..... 5

The dermis ................................................................................................... 10

The dermo-epidermal junction ..................................................................... 11

When the barrier is breached .......................................................................... 13

Non-healing wounds .................................................................................... 18

Wound treatment - dressings, skin substitutes and scaffolds ......................... 20

Skin substitutes and scaffolds ..................................................................... 22

Gelatin microcarriers ................................................................................... 25

Modelling skin and wound healing ................................................................... 26

Human full thickness skin in vitro ................................................................ 29

Skin, wound healing and reciprocity ............................................................ 31

AIMS OF THESIS ................................................................................................ 35

MATERIAL AND METHODS ............................................................................... 37

Primary cell culture and media .................................................................... 37

Microcarriers and spinner flask culture ........................................................ 39

Viability assay .............................................................................................. 40

Migration assay ............................................................................................ 41

Quantitative real-time polymerase chain reaction ....................................... 42

Human in vitro wound healing model .......................................................... 43

Non-occlusive topical exposure ................................................................... 44

Paraffin embedding and sectioning ............................................................. 45

Cryosectioning ............................................................................................. 46

Haematoxylin and eosin staining ................................................................. 46

Immunohistochemistry ................................................................................. 48

Carboxyfluorescein hydroxysuccinimidyl ester (CFSE) staining ................. 49

Flow cytometry ............................................................................................. 50

Fluorescence microscopy and confocal microscopy ................................... 51

Statistical analyses ...................................................................................... 52

Ethical considerations .................................................................................. 53

SUMMARY OF PAPERS ..................................................................................... 55

Paper I ............................................................................................................. 55

Paper II ............................................................................................................ 59

Paper III ........................................................................................................... 63

Paper IV ........................................................................................................... 67

CONCLUDING REMARKS .................................................................................. 73

ACKNOWLEDGEMENTS .................................................................................... 74

REFERENCES .................................................................................................... 76

1

LIST OF PAPERS

This thesis is based on the following papers, which are referred to in the text by

their Roman numerals:

Paper I

Susanna Lönnqvist, Jonathan Rakar, Kristina Briheim, Gunnar Kratz

Biodegradable gelatin microcarriers facilitate re-epithelialization of human

cutaneous wounds – an in vitro study in human skin

PLoS One 2015 Jun 10;10(6)

Paper II

Susanna Lönnqvist, Maria Karlsson, Gunnar Kratz

Tracing transplanted human keratinocytes and melanocytes with

carboxyfluorescein hydroxysuccinimidyl ester (CFSE) staining

Manuscript

Paper III

Susanna Lönnqvist, Peter Emanuelsson, Gunnar Kratz

Influence of acidic pH on keratinocyte function and re-epithelialisation of human

in vitro wounds

Journal of Plastic Surgery and Hand Surgery 2015 Jun 7:1-7

Paper IV

Susanna Lönnqvist, Kristina Briheim, Gunnar Kratz

Non-occlusive topical exposure of human skin in vitro as model for cytotoxicity

testing of irritant compounds

Toxicology Mechanisms and Methods 2015 Oct 8:1-6

2

Publication by the author not included in the thesis:

Kristin M. Persson, Susanna Lönnqvist, Klas Tyrbrandt, Roger Gabrielsson,

David Nilsson, Gunnar Kratz, Magnus Berggren

Matrix-Addressing of an Electronic Surface Switch Based on a Conjugated

Polyelectrolyte for Cell Sorting

Advanced Functional Materials 2015 Oct 21

Johan Junker, Susanna Lönnqvist, Jonathan Rakar, Lisa Karlsson, Magnus

Grenegård, Gunnar Kratz

Differentiation of human dermal fibroblasts towards endothelial cells

Differentiation 2013 Feb;85(3):67-77

Jonathan Rakar, Susanna Lönnqvist, Pehr Sommar, Johan Junker, Gunnar Kratz

Interpreted gene expression of human dermal fibroblasts after adipo-, chondro-,

and osteogenic phenotype shifts

Differentiation 2012 Nov;84(4):305-13

3

Abbreviations

α-SMA α-smooth muscle actin

ANOVA analysis of variance

bFGF basic fibroblast growth factor

BPE bovine pituitary extract

BSA bovine serum albumin

Ca2+ calcium cation

cDNA complementary deoxyribonucleic acid

CFSE carboxyfluorescein hydroxysuccinimidyl ester

CLDN1 claudin 1

COL collagen

Ct cycle threshold

DAPI 4’, 6-diamino-2-phenylindole

dbcAMP N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate

DMEM Dulbecco’s Modified Eagle Medium

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

ECM extracellular matrix

ECVAM European centre for validation of alternative methods

EDC epidermal differentiation complex

EDTA ethylenediamine tetra acetic acid

EGF epidermal growth factor

FAK focal adhesion kinase

FCS foetal calf serum

FM fibroblast medium

GM-CSF granulocyte-macrophage colony stimulating factor

H&E haematoxylin and eosin staining

HMBS hydroxymethylbilane synthase

HSP27 heat shock protein 27

HSPB1 heat shock 27 kDa protein 1

ICAM-1 intercellular adhesion molecule 1

IFN- ɣ interferon ɣ

IL interleukin

KER keratinocyte

KGF keratinocyte growth factor

Ki-67 nuclear antigen Ki-67

4

KLK kallikrein-related peptidase

KRT keratin

KSFM keratinocyte serum free medium

MEL melanocyte

MGM melanocyte growth medium

MHC major histocompatibility complex

MMP matrix metalloproteinase

mRNA messenger ribonucleic acid

MTT 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide

OCT optimal cutting temperature compound

PBS phosphate buffered saline

PC-1 PC-1TM supplement

PCL polycaprolactone

PDGF platelet derived growth factor

PFA paraformaldehyde

PGA polyglycolic acid

PLA polylactic acid

PTK2 protein tyrosine kinase 2

qRT-PCR quantitative real time polymerase chain reaction

RGD arginine-glycine-aspartate tripeptide

RMP revolutions per minute

RNA ribonucleic acid

S100A S100 calcium binding protein A family

SDS sodium dodecyl sulphate

TGF- α transforming growth factor α

TGF- β transforming growth factor β

TIMP1 tissue inhibitor of matrix metalloproteinase 1

TNF- α tumour necrosis factor α

UV ultraviolet

VEGF vascular endothelial growth factor

ZO-1 zona occludens 1

5

INTRODUCTION

The skin constitutes a physical and molecular barrier against dehydration,

pathogens and toxins. Stratum corneum, the outermost layer of the skin, forms a

lipophilic barrier and together with a band of tight junctions and the innate immune

system, comprise the first line of defence against the strains of the outside world.

The layered structure compartmentalises the functions of the skin. The uppermost

layer, the epidermis, forms the first line of barrier. The dermis underneath provides

mechanical strength and possesses barrier, sensory and immune functions. The

hypodermis forms the structural integrity of the tissue and provides vascularisation

(Figure 1).

The structure of the epidermis is linked to the life cycle of the

keratinocyte

The epidermis is a keratinised stratified squamous epithelium composed

predominantly of keratinocytes as well as melanocytes, Langerhans’ cells and

Merkel cells. The epidermis consists of four layers: stratum basale with

proliferative keratinocytes, stratum spinosum, stratum granulosum, and the

stratum corneum (1). An additional layer between granulosum and corneum called

stratum lucidum is present in areas of skin highly subjected to friction such as the

palms and soles (2). The keratinocytes of the epidermis stem from epidermal stem

cells which are present in the stratum basale. By dividing they give rise to the

transit amplifying cell compartment that ultimately undergoes terminal

differentiation and cell death to form the stratum corneum of densely packed cell

membranes of keratinocytes (3). Keratinocytes express specific sets of keratins

during their passage through the epidermis. Keratins are elastic fibrous proteins

that form cytoplasmic intermediate filaments and are expressed in heterodimeric

pairs with one acidic (type I) and one basic (type II) keratin (4). Basal keratinocytes

express keratin 5 (type II) and keratin 14 (type I), and differentiating keratinocytes

6

express keratin 1, 2 (type II), and 10 (type I) (5). The keratin filament network

anchors keratinocytes to the basement membrane through hemidesmosomes in

stratum basale (6) and to adjacent keratinocytes through desmosomes,

intercellular junction complexes associated with intermediate filaments (7), in all

layers of epidermis (8). The stratum granulosum (Figure 1) consists of three layers

of cells and is characterised by the presence of keratohyalin granules consisting of

keratin binding proteins, such as filaggrin and loricrin. Together with mainly

keratin 1 and 10, the proteins form a dense network of keratin filaments bundles

(1). A strand of tight junctions is present in the second layer of cells in the stratum

granulosum. This structure can be found on the apical side of most sheets of simple

epithelium in vertebrates and functions as a sealing to control the paracellular

pathways (9) for ions, small molecules and water (10). In the epidermis the cell-

cell junctions consist of different transmembrane proteins like claudins (11) and

cytosolic plaque proteins like zona occludens (ZO) proteins, out of which claudin

1 (CLDN1) has been shown to contribute strongly to the integrity of the junctional

complexes (10). Knock-out mice for CLDN1 die within the first day after birth due

to transepithelial water loss (12). The stratum granulosum is a transitional zone of

the epidermis where the separation of metabolically active layers and the dead

stratum corneum takes place.

The stratification of the epidermis is tightly connected to the life cycle of the

keratinocyte. The keratinocytes of the uppermost layer of the stratum granulosum

undergo terminal differentiation and a form of programmed cell death called

cornification. The end product is the formation of the stratum corneum composed

of 10-20 layers of enucleated cells forming a supracellular interconnected structure

consisting of corneocytes (13). The formation of the stratum corneum takes place

in three major steps: the replacement of intracellular contents of the keratinocytes

by a proteinaceous cytoskeleton, the formation of the cornified envelope and the

secretion of extracellular lipids. At the transition zone of the stratum granulosum

7

Figure 1. (A) Haematoxylin and eosin (H&E) staining of human skin with stratified,

cornified epidermis and papillary and reticular dermis. (B) Schematic

representation of the layers and cells of the epidermis. (C) H&E staining of hair

follicle. (D) Schematic drawing of human skin with superficial epidermis, dermis

with hair follicle and vessels, and subcutaneous fat of the hypodermis. Images

captured or drawn by author, except (B) based on Servier Medical Art (licensed

under Creative Commons).

8

keratinocytes undergo enucleation and morphologically change into flat polygonal

cells. The organelles and the keratohyalin granules are degraded. The mechanisms

of nuclear degradation and the following disassembly of the organelles are not well

understood (14). The dephosphorylation of profilaggrin has been proposed as an

initiating step in disassembling the keratin and keratin binding proteins (1, 15).

The transition from residing in the stratum granulosum to partake in the formation

of the stratum corneum, is initiated by an increase in intracellular Ca2+ (16), which

has been shown to induce in vitro differentiation of keratinocytes as well (17). In

addition, caspase 14 is involved in the process and is activated in the granular layer

and contributes to the degradation of filaggrin (15). The keratinocytes form

cornified envelopes where the plasma membranes are replaced by protein products

of the epidermal differentiation complex (EDC) genes that are highly crosslinked

by calcium activated transglutaminases specifically expressed in the cornifying

layers (18). The EDC locus on chromosome 1q21 contains genes like loricrin,

involucrin and the S100A family, and their expression is used as late maturation

markers of the epidermis. The dense assembly of the transglutaminase crosslinked

proteins is essential for the barrier function of the skin (19). The final stages of the

life cycle of granular keratinocytes include the formation of the cornified envelope

and the secretion of the lamellar granules, membrane bound cytoplasmic

organelles found in stratum spinosum and granulosum. The lamellar granules, or

lamellar bodies, fuse with the plasma membrane and thereby contribute to the

hydration of the epidermis by secreting their contents into the extracellular space.

The ceramides, free fatty acids and cholesterol of the lamellar granules seal the

superficial granular layers and are responsible for the hydrophobic barrier of the

epidermis (20).

The cornified keratinocytes of the stratum corneum are joined together through

corneodesmosomes, an adhesion complex similar to the desmosome, but

characterised by the presence of a unique extracellular component known as

9

corneodesmosin (21). Produced by keratinocytes and secreted by granular

keratinocytes through the lamellar granule pathway, it is localised in the

extracellular structures of the corneodesmosomes and cross-linked to the cornified

cell envelopes (20). Corneodesmosin is gradually proteolysed under transition in

the stratum corneum and ultimately cleaved by kallikrein-related peptidases

(KLKs) and cathepsins (22). This leads to the desquamation of the epidermal

keratinocyte, the final step in the keratinocyte’s role as the structural component

of the barrier of the epidermis (23).

Other cell types in the epidermis include the pigment producing melanocytes,

Langerhans’ cells and Merkel cells. Melanocytes are located in the stratum basale

(24) and make up approximately 5 % of the cells of the epidermis (25). One

epidermal-melanin unit consists of one dendritic melanocyte and an average of 36

keratinocytes (26). Once formed, melanin containing granules, called

melanosomes, are translocated to the dendritic tips of the melanocytes by active

transport (27). The melanosomes are transferred to keratinocytes by shedding

vesicle transport and the melanosomes are trapped by microvilli on keratinocytes

and incorporated into the cytosol. Once inside the cell, the melanosomes are

dispersed with the ultimate goal to form a perinuclear cap in the keratinocytes to

protect DNA from UV-light induced damage (28).

The only immune cells resident in the epidermis are the Langerhans’ cells. As

immature dendritic cells, they phagocytose actively in stratum spinosum. In the

case of an injury and infection Langerhans’ cells migrate to the peripheral lymph

nodes, loose the antigen processing capabilities but upregulate major

histocompatibility complex (MHC) and present antigens at a high level (29).

Merkel cells are mechanosensory cells located in the basal layer and form synapse-

like connections to somatosensory afferents in the dermis where they convey touch

10

and pressure sensations (30). Merkel cells have been shown to be essential for the

fine tuning of the pressure sensation which enables light touch discrimination of

textures and edges (31).

The dermis

The connective tissue that constitutes the dermis is produced by dermal fibroblasts.

Additional cell types present are macrophages and adipocytes. The dermis

harbours nerves, glands, hair follicles and blood vessels that supply the skin and

participate in thermal regulation. The most abundant proteins of the extracellular

matrix (ECM) of connective tissues, and the whole human body, are collagens

(32). By forming trimeric triplehelices that are organised into fibrils, the network

formed by collagens has a high structural integrity that accounts for the toughness

of skin. The fibril forming collagens I and III are present in the dermis. Basement

membranes, and the stratum basale, contain collagen IV, which connects to

collagens in the dermis via the anchoring fibril collagen VII (33). The elastic

properties of the dermis are on account of the presence of micro fibrils of fibrillin

with a core of cross-linked elastin. The elastic fibres provide compliance and recoil

properties to the tissue and complements the collagen fibrils to form a resilient

tissue construction (34). A dermal ECM protein with high importance for cell

adhesion and migration is fibronectin (35, 36). The integrin binding RGD motif

(arginine-glycine-aspartate tripeptide) was discovered in fibronectin. Fibronectin

is an example of how ECM proteins can bind other ECM constituents, growth

factors, receptors and adhesion proteins (37), and contribute to both structure and

function of the ECM. Integrins are heterodimeric transmembrane receptors that

can arrange into 24 different combinations and have high affinities for molecules

in the ECM (38). The extrafibrillar matrix of the ECM beyond the fundamental

structural proteins consists of glycosaminoglycans like hyaluronic acid, and

proteoglycans. Proteoglycans contribute to the resistance to compression of the

11

dermis by being highly hydrophilic due to the positively charged polysaccharide

chains (39, 40).

The structural division of the dermis in two layers is based on the packing of the

connective tissue. The most superficial part of the dermis, the papillary dermis, is

characterised by areolar connective tissue organised in dermal papillae forming

projections into the epidermal side. Tactile receptors, the Meissner corpuscles (41),

and free nerve endings are present in the papillae that are responsible for the

conduction of sensations of e.g. pain. Somatosensory afferents from the reticular

dermis underneath connects to Merkel cells in the basal layer of the epidermis

through Merkel discs located in conjunction with the basement membrane,

forming Merkel cell-neurite complexes making up a direct contact between the

two tissue compartments (30, 42). The reticular dermis is a dense network of

collagens and coarse elastic fibres making up the bulk of the skin and providing

for its integrity and extensibility. Interspersed in the spaces between the connective

tissue bundles are adipocytes, glands, nerves and the hair follicles. Epidermal cells

are present in two of the structures of the hair follicle: in the matrix surrounding

the connective tissue papilla and in the bulge of the hair follicle (Figure 1).

Keratinocytes and melanocytes are resident in the matrix of the hair follicle and

the origin of the epithelial cells is the epidermal stem cells of the bulge of the hair

follicle (43). The bulge cells only contribute to the structure of the epidermis in the

case of re-epithelialisation of a wound where the cells respond quickly to damage

but give rise to a transit amplifying population that later is replaced by strictly

epidermal keratinocytes (44).

The dermo-epidermal junction

The epidermis and dermis are separated by a basement membrane (Figure 1) (45).

The membrane forms the physical division between the tissue compartments,

restricts molecular transport between them and dictates polarity of the basal

12

keratinocytes which is of importance for the directionality of the differentiation

and maturation of the epidermis (46). The dermo-epidermal junction is unique

within basement membranes in its structure due to the presence of anchoring

complexes. These structures establish integrin-mediated connections to link basal

keratinocytes to the basement membrane. Integrin combinations expressed by

basal keratinocytes are α2β1, α3β1, and α6β4, out of which α4β6 is strictly

expressed on the basal side of the keratinocytes, opposing the basement membrane

(47) and exclusively expressed in the hemidesmosomes in mature epidermis (48).

The anchoring complex proteins are the link between the cytokeratin intermediate

filaments of the basal keratinocytes and the basement membrane (Figure 2). The

anchoring filaments are 2-4 nm in diameter and predominantly consist of laminin

5. Laminins are heterotrimeric basement membrane proteins composed of α, β and

γ chains. Two nomenclatures are used today: laminin 5 was the fifth trimer

composition discovered, but new nomenclature names laminins after chain

numbers rendering laminin 5 and laminin 322 the same protein (49). Laminin 5

connects the hemidesmosomes through integrin α6β4 to other laminins and

collagen VII in the basement membrane compartment. Collagen VII in turn links

the fibrous matrix elements (collagens I, III and V and fibrillins) and thereby

anchors the complete complex to the papillary dermis. The anchoring complex

Figure 2. Organisation of

the basement membrane

with connections to the

basal keratinocyte via

integrins, and the

collagens in papillary

dermis. COL: collagen.

Adapted from Uitto et al.

2001 31

13

system provides the structural integrity needed for the frictional forces that skin is

subjected to (47, 50).

When the barrier is breached

When wounding occurs, a myriad of processes are initiated to restore the barrier

of the skin. Wound healing is a dynamic process involving all cell types in the

skin, soluble factors and the ECM (51). A broad subdivision of the process is the

inflammatory phase, proliferative phase and tissue remodelling phase (Figure 3).

The inflammatory phase is a direct response to the disruption of the tissue and the

resident blood vessels. The blood clot formation achieves haemostasis and

provides the cells in the wound with a provisional ECM. Platelets and injured cells

start secreting mediators that recruit monocytes, fibroblasts and leukocytes to the

site (52, 53). Infiltrating neutrophils remove debris and bacteria from the site and

monocytes are recruited by ECM fragments and transforming growth factor β

(TGF-β). Once activated, the macrophages release platelet derived growth factor

(PDGF) (54) and vascular endothelial growth factor (VEGF), essential for

formation of the granulation tissue (55). Granulation tissue is the provisional ECM

formed during the wound healing process by major rearrangement of the ECM and

that will eventually form the novel stroma (56, 57). Other monocyte and

macrophage derived growth factors in the early inflammatory stage of wound

healing are TGF-α, interleukin 1 (IL-1) and TGF-β (58) (Figure 3 B).

Epidermal keratinocytes are presented with an alternative pathway to

differentiation upon injury, the pathway of activation. The activation of

keratinocytes to hyperproliferative and migratory cells (59) is affected by

extracellular stimuli and signals, and characterised by differential expression of

keratins. The activation of keratinocytes leads to proliferation, migration,

upregulation of cell surface receptors and production of basal membrane

14

components, all important for the re-epithelialisation process. Re-epithelialisation

commences within hours of injury (58, 60, 61). Activated keratinocytes express

keratins 6, 16 and 17 (5, 62). The accumulation of keratin 6 and 16 in wound edge

keratinocytes coincides with the rearrangement of keratin 5 and 14 filaments

changing from pancytoplasmic to concentrated at the edge of the cell, directed

away from the migrational leading edge. The major cytoarchitectural changes

precede the migration of keratinocytes out into the wound bed which also requires

changes in the structure and number of desmosomes (63). The initiation of re-

epithelialisation also requires downregulation of hemidesmosomal links between

the epidermis and the basement membrane to allow lateral movement of activated

keratinocytes (58, 64).

Epidermal keratinocytes are on constant stand-by for responding to attacks on the

barrier. Sequestered in the cytoplasm, keratinocytes carry IL-1, both α and β forms

(65). IL-1 is the key initiator of keratinocyte activation and is rapidly processed

and released after injury (66). Keratinocytes express both types of IL-1 receptors

and their antagonist making them highly responsive and sensitive to the effects of

IL-1. IL-1 functions as an autocrine signal for keratinocytes to enhance the

activation cycle (67) (Figure 4). IL-1 plays a crucial role for the paracrine

signalling as it activates endothelial cells, fibroblasts and lymphocytes. IL-1 is a

chemoattractant for lymphocytes and together with activation of endothelial cells

and induction of selectin expression, IL-1 initiates lymphocyte extravasation (68).

Cytokines and growth factors induced by IL-1 in keratinocytes include

granulocyte-macrophage colony stimulating factor (GM-CSF) (69), tumour

necrosis factor α (TNF- α) and transforming growth factor α (TGF-α), as well as

IL-1. TNF-α has been shown to maintain the activation of keratinocytes (70) and

elevated levels have been found in conditions like irritant contact dermatitis and

infections (71). The maintained activation leads to production of several cytokines

15

like IL-3, 6 and 8, which contribute to the paracrine signalling of keratinocytes in

the wound healing environment (5, 72).

Figure 3. (A) The overlapping stages of the wound healing process at different times

after wounding. (B) Schematic overview of the inflammatory phase of wound

healing with involved cell types and soluble factors. KGF: keratinocyte growth

factor, PDGF: platelet derived growth factor, TGF: transforming growth factor,

TNF: tumour necrosis factor, VEGF: vascular endothelial growth factor. (A)

adapted from Komosinska-Vassev et al. 2014 122 (B) from Singer et al. 1999 32

16

The signals form a positive feedback loop with increased production and

upregulation of cell surface receptors and intercellular adhesion molecule 1

(ICAM-1) and integrins, essential for the migration of keratinocytes when the re-

epithelialisation of the wound starts (5). The integrins expressed on activated

keratinocytes allow interaction with fibrinogen and collagen type I which is

present in the wound edge and interwoven with the fibrin in the blood clot. As the

keratinocytes migrate to cover the wound bed they are guided by the array of

integrins they express (73). The migrating keratinocytes create an optimal

environment for migration in additional ways: they contribute to the degradation

of the ECM, which is necessary for migration to take place, by producing

collagenase and matrix metalloproteinases (MMPs) (74, 75). The new stroma,

referred to as granulation tissue, starts filling out the wound bed after day four in

the wound healing process. Produced by fibroblasts, the new connective tissue is

rich in capillaries (58, 76). Both angiogenesis and vasculogenesis, with recruitment

of bone marrow-derived progenitor cells, contribute to the vascularisation of the

granulation tissue in the proliferative phase of wound healing (77). The formation

of a vascular network is pivotal for tissue repair in terms of oxygenation and

nutrient supply (78). Fibroblasts and macrophages continue moving into the

wound as the provisional matrix is formed. Macrophages sustain the growth factor

production to induce proliferation of all surrounding cell types and fibroblasts

produce and deposit ECM rich in fibrin, fibrinogen and hyaluronic acid (51, 79).

The provisional wound healing matrix and granulation tissue that enables

migration of keratinocytes and fibroblasts, and oxygenation, is gradually replaced

by fibroblasts that start depositing a collagenous matrix (80).

A contained proliferative burst is required to sustain the re-epithelialisation

process. The keratinocytes behind the actively migrating wound edge start

proliferating one to two days after injury (51). The inducing factors have not been

established. The free edge effect has been proposed as one of the mechanisms

17

where the absence of neighbouring cell, in combination with growth factors,

signals both migration and proliferation (58). As re-epithelialisation is completing,

the basement membrane components collagen type IV and laminin reappear and

fibronectin and fibrinogen are downregulated (57). At the late stage of re-

epithelialisation where contraction of the restored basal membrane is required the

activated keratinocytes obtain a contractile phenotype that is characterised by

expression of keratin 17 (5, 81). Keratin 17 is present in basal layers of

pseudostratified epithelia and myoepithelial cells, which have in common that they

contract or change shape. The expression of keratin 17 in epidermis is restricted to

the activated contractile keratinocyte and directly induced by interferon ɣ (IFN-ɣ)

(38, 82). IFN-ɣ is an autocrine signal for lymphocytes and a paracrine signal for

keratinocytes conveying the signal of the late inflammatory response (83). A de-

activation signal in the form of TGF-β is expressed at this time in the wound

healing process by dermal fibroblasts. TGF-β suppresses keratinocyte cell

proliferation and directly

induces basal-specific

expression of keratin 5

and 14 (84). The effect of

TGF-β on keratinocyte

activity has been shown

to be reversible and does

not lead to terminal

differentiation but

reduces the growth rate

back to a normal basal

level (85, 86).

The remodelling phase of the wound healing process is the long process of ECM

reorganisation at the site of injury that will eventually lead to the formation of a

Figure 4. Keratin expression in keratinocytes and

factors involved in the keratinocyte activation

cycle. Adapted from Blumenberg et al. 2001 5

18

fibrous scar. The maturation of a scar can take years to complete. The initiation is

characterised by the apoptosis of granulation tissue fibroblasts, a process for which

no inducing signal is known (87, 88). With this, the formation of granulation tissue

is stopped and the collagen type III that was deposited during the inflammatory

phase is degraded. It is replaced by type I collagen oriented in small parallel

bundles, different to the basket weave organisation of normal dermal collagen.

This orientation of collagen I is responsible of the different texture and appearance

of a cutaneous scar (89). During the remodelling phase, fibroblasts in the vicinity

of the wounded area obtain a contractile phenotype referred to as myofibroblast

(90). Myofibroblasts contribute to the closing of the wound by contracting the

remodelling ECM and diminishing the wound area. Mature myofibroblasts express

α-smooth muscle actin (α-SMA) stress fibres. The most well accepted inducer of

myofibroblast activation is TGF-β1 (91, 92). TGF-β1 and 2 are key regulators of

the inflammatory phase of wound healing, and play a role in promoting fibrous

scar formation (93, 94). Excessive expression of TGF-β1 and 2 has been shown to

promote aberrant scarring, and dysregulation of the remodelling phase is

manifested as hypertrophic or keloid scarring. In both cases the deposition of scar

tissue is extensive, forming raised scars. Hypertrophic scars remain within the

boundaries of the original injury whereas keloids grow beyond (95, 96).

Myofibroblasts are also involved in the pathophysiology behind hypertrophic

scars, especially following burn wounds, where myofibroblasts are numerous and

heavily contract the scar. Scar contraction can be painful and disabling, and

persists as a major complication following burns (91).

Non-healing wounds

Patients suffering from non-healing, or chronic, wounds are a large patient group

in today’s health care systems (97). The complications impose morbidity and

mortality on mostly the elderly patient group and bring about suffering for long

19

term patients with pain and substantial effects on quality of life (98). Treatment

times are often prolonged and the risk of recurrence is high, and it is estimated that

2 % of health care budgets in Scandinavia are consumed by costs associated with

chronic wounds (99). A prevalence estimation of foot and leg ulcers on the

Swedish general population is 0.12-0.2 % (100, 101).

The term chronic wound includes all wounds that fail to heal and include venous

and arterial leg ulcers, diabetic foot ulcers and pressure ulcers (102-104). Cancer

and radiotherapy can also lead to impaired healing (105), and all these

preconditioning states are more prevalent in older adults. This patient group also

undergoes surgery more often which increases the risk for chronic wound

complications. Conditions that affect the vascular system affect the vasoregulation

of the microcirculation of the skin which can lead to hypoperfusion (106). This

reflects in changes in the inflammatory response in the skin during wound healing,

low oxygen tension and poor nutrient delivery (107, 108). The resident cells in the

chronic wound are also characterised by decreased proliferation rates and

morphology resembling senescent cells. Fibroblasts isolated from venous leg

ulcers have been shown to have a reduced response to PDGF and lower expression

of TGF-β receptors compared with normal dermal fibroblasts. These are similar

responses seen in fibroblast exposed to hypoxia, indicating that chronic wounds

are hypoxic (107, 109, 110).

Chronic wounds are often characterised by being halted in the inflammatory phase

of the wound healing process (111). An increase of cellular infiltrates that is

persistent in a chronic wound leads to the prolonged presence of neutrophils and

macrophages. This leads to the dysregulation of some of the key regulators of the

inflammatory phase: IL-1β and TNF-α have been shown to prolong the

inflammatory phase and delay wound healing (112, 113). Venous stasis ulcers are

the most prevalent lower limb ulceration (114). Venous hypertension is caused by

20

an obstruction or reflux, and the venous insufficiency resulting from it leads to

dilation of capillaries and leakage of plasma proteins, fibrin deposition and a lower

oxygen tension in the tissue. The effects are ischemia and hypoxia which leads to

cell death and ulceration (115). The venous leg ulcers have many underlying

pathogeneses but have in common the complications that are caused by prolonged

inflammation times. This often leads to substantial microbial colonisation, a

system that once established can sustain itself (116, 117). The chronic ulcers also

have an aberrant expression of MMPs due to the elevated levels of IL-β1 and TNF-

α (112) and their locally expressed inhibitors (118) and inflammatory cytokines

which degrade the ECM and vascular walls in the healing wound environment. In

the case of diabetic foot ulcers, oxidative stress has been identified as one of the

key problems. The stagnation in the inflammatory phase leads to continuous

infiltration of neutrophils that release free radicals and inflammatory mediators,

which causes cytotoxic effects on the surrounding tissues and delays wound

closure (116, 119).

Wound treatment - dressings, skin substitutes and scaffolds

There are several types of different wound dressings for the treatment of non-

healing wounds aimed to enhance wound healing. The foremost contribution of

coverage of a wound with a dressing is the barrier effect it has and the maintenance

of a moist wound healing environment (120). Providing a temporary barrier to

reduce infection and minimise necrosis is the first line of action. Open wounds are

frequently contaminated and chronic wounds are easily colonised with bacteria

that can start spreading into the tissue, and infect the wound. The poor

vascularisation and devitalised tissue that offers a favourable milieu for

microorganisms contribute to the risk of infection. Debridement, cleansing and

coverage are essential to minimise colonisation and prevent severe infection (121,

122). Moist or wet treatment of wounds has been shown to enhance the wound

21

healing process seen by a promotion of re-epithelialisation and reduction of

inflammation. Before the 1960s and the introduction of the idea of moist wound

healing, wounds were often treated dry with the notion that infection would be

easier to combat in a dry environment (123), until Winter et al. showed accelerated

re-epithelialisation of wounds on pigs treated with occlusive dressings (124). In

1963 Hinman and Maibach showed occlusion of experimental wounds to be

beneficial when treating human wounds (123). Since then, moist dressings are

standard care for chronic ulcerations (125). There is a vast range of dressings with

a multitude of mechanisms of actions including incorporated antibiotics and

growth factors (126-134). Dressings and occlusion have also been utilised to

modulate components of the local wound environment, like pH value. With limited

clinical evidence of enhanced wound healing and difficult interpretation of clinical

trials with few randomised controlled trials available, simple dressings can be

considered as a protective barrier to provide a suitable wound healing environment

(111). An optimal wound dressing should fulfil certain criteria: provide hydration

but remove exudate and be impermeable to microbes, but allow gas diffusion. The

dressing should not release unfavourable agents or fibres, and not harm the

periwound area when removed. Finally, the product should be easy to use and cost

effective. Few dressings meet all requirements (134).

Efforts to improve wound healing are of course not restricted to chronic wound

problematics. Burn patients are a large patient group with acute need for barrier

restoration. Immediate measures taken are removal of necrotic tissue and wound

coverage and subsequent autologous split or full thickness skin grafting with the

goals of minimising bacterial colonisation to avoid sepsis, and to preserve as much

of the unharmed tissue as possible (135). The problem with large burns is the

scarcity of healthy donor sites for a graft, and donor site morbidity due to the poor

general status of the patients. Cultured epidermal autografts were introduced in the

1980s (136). Keratinocyte sheets can be expanded and stratified in culture before

22

reintroducing them to the patient (137). A drawback is the long culture time

required (135). To solve disadvantages of using epidermal autografts, autologous

keratinocytes can be transplanted before reaching a confluent state as a single cell

suspension, often with fibrin sealant (138, 139). The keratinocytes retain their

proliferative state and contribute to re-epithelialisation (140). The greatest

advantages are the shortening of culture times and the ease of handling. When

transplanting subconfluent keratinocytes, the total cell viability is also higher

compared with epidermal autografts (141). Autologous transplantation of

keratinocytes, as epidermal autografts or in suspension, is a life saving measure

that has improved burn wound care substantially, however the methodology does

not support regeneration of the complete skin. The dermal component is still

lacking, and transplantation sites are left with a thin, brittle skin that lacks

thermoregulation, hair follicles and has sensory deficiencies (142).

Skin substitutes and scaffolds

There are several approaches to substituting the dermis, ranging from applying a

scaffold for resident cells to populate as a way to induce guided tissue regeneration,

to engineered constructs with living cell components (143-146). Initial tissue

engineering efforts to replacing tissues utilised materials that were inert, with the

notion that the material could not degrade and harm the host (147). Later

development has led to the use of degradable scaffolds that aims to deliver cells,

genes and/or proteins to the harmed tissue and gradually degrade, leaving space to

the regenerated tissue. The ultimate scaffold will support tissue regeneration by

preserving the tissue volume, and guide ingrowth and regeneration of the native

tissue (148). The degradation rate of the scaffold should match the rate by which

the new tissue is regenerated at the site of implantation and the scaffold should

provide structural integrity to the damaged tissue for a certain period of time before

adapting to the environment. Tissue guidance is allowed by an open scaffold

system, where eventually the complete scaffold will be degraded or completely

23

integrated with the host (149). Scaffold design in terms of choice of material and

porosity, besides the obvious biocompatibility, plays a large part in the suitability

of the material for tissue engineering purposes. The target application dictates the

requirements set on the material of choice, but in general terms the selection is

based on the physical, mass transport and interaction properties (150). Moreover,

mechanical strength of the material, gelation and diffusion properties need to be

considered. Out of mass transport properties, diffusion is the most important one

to provide oxygenation into the scaffold, and will depend on both size of the

molecule and charge interactions with the scaffold material (151-153). The

mechanical strength dictates the space filling properties of the material and the

degradation rate that is crucial for optimal performance. The rigidity of the scaffold

is also important in terms of mechanical input to the native cells (154-156).

Porosity coheres with the gelation of a material and its subsequent topography and

biological properties. As the scaffold is to be implanted, it should promote cellular

function in order to work as a guiding scaffold. Adherence, proliferation and

differentiation of cells should be supported (151).

Hydrogels have been extensively investigated in the field of tissue engineering of

skin. The structure of gels with high wettability (≥30 % water content by weight)

(151, 157) is similar to macromolecular components of connective tissue and

suitable for implantation due to the low strain on native tissue they exert. Both

synthetic and biological degradable polymers are utilised for hydrogel production.

The most applied synthetic polymers include polylactic acid (PLA), polyglycolic

acid (PGA) and polycaprolactone (PCL) (149) . Synthetic polymer scaffolds with

the aim to restore the epidermis have largely been unsuccessful, mainly due to the

low cellular recognition and compatibility (158). Efforts are being made to

combine the synthetic polymers with biopolymers to make use of preferable

properties from both. Synthetic polymers have the advantage of fabrication where

desirable physical features can be added (159) and the natural biopolymers carry a

24

biocompatibility that allows adherence of cells carrying adhesion molecules for

natural components (151). Degradable biopolymers used in scaffolds for

regeneration of skin explored today are mostly based on agarose, alginate,

chitosan, fibrin, hyaluronic acid, gelatin and collagen.

Acellular constructs are designed to mimic ECM components and to provide a

dermal scaffold to support dermal regeneration (160). Early studies on materials

for wound dressings and skin tissue engineering understandably focused on

collagen, and being the main component of the ECM there are several collagen

based dressings on the market today in the form of gels, sheets and sponges. For

tissue guidance, acellular dermal allografts are available commercially today. One

of the most established is AlloDerm (LifeCell, Branchburg, NJ) (159) derived from

human cadaveric skin that is acellularised, lyophilised and glycerolised. It

naturally resembles the native dermal tissue (161). Today, AlloDerm is primarily

applied to partial and full thickness burns, but also used for soft tissue replacement

and reconstruction of abdominal wall defects (162). Cellular dermal allografts are

the next level of complexity of skin substitutes, where a bovine or porcine collagen

matrix is seeded with neonatal fibroblasts that remain viable for a limited time, but

exert positive effects for regeneration of the dermal tissue (163). These constructs

are focused on the aiding in regeneration of a dermis, but efforts are being made

to provide constructs with both dermal and epidermal components. Integra (Integra

LifeSciences, Plainsboro, NJ) is a synthetic bilayer product. It is a composite of

collagen and chondroitin-6-sulfate of bovine origin with a silicone cover sheet

(164). The matrix is engineered to have pores in the range of 20-50 µm. Three to

four weeks after implantation for tissue guidance for dermal cells, the silastic cover

is removed and an epidermal graft can be placed on the treated area. Both

AlloDerm and Integra have been largely successful in providing resident cells with

a space filling guiding scaffold, and more importantly in preservation of tissue

volume after large trauma or burns. Nonetheless, they are hampered by high costs

25

and the need for several surgeries if an epidermal component is to be added. The

prototypes for human allogenic skin substitutes including both dermal and

epidermal components are Apligraf (Organogenesis, Canton, MA) (165) and

OrCel (Ortec Intl.Inc., NY) (166). The dermal matrix is a bovine type I collagen

gel with embedded neonatal fibroblasts. Neonatal keratinocytes are seeded on top

of the constructs. These types of constructs are the most advanced products for

treatment of wounds and present the closest resemblance to native tissue. The use

of cellular allogenic composites is restricted due to high costs compared with

conventional treatment strategies, a statement which in turn is debated when taking

into account the quality of healing and loss of recurrence and later contacts with

the health care system for the patients (162) .

Gelatin microcarriers

Bilayer constructs and dermal allografts

represent scaffolding that are of a rigid nature

and present a need for prefabrication.

CultiSpher-S gelatin porous microcarriers

(Percell Biolytica, Sweden) are degradable

microcarriers ranging in size from 70 – 170 µm

in diameter (Figure 5). The carriers are used in

suspension, enabling the use of the scaffold for

any type or size of lesion. CultiSpher-S consist

of porcine type A gelatin that is highly

crosslinked. Gelatin is a derivative of collagen

and therefore highly biocompatible and suitable as a biomaterial for guided

regeneration of skin. The triple helix structure unique for collagen can be broken

to obtain single chains, i.e. gelatin. Type A gelatin is obtained by acidic treatment

of collagen (167). Gelatin has been widely investigated in different biomedical

applications due to its natural origin, biocompatibility and degradability (168).

Figure 5. CultiSpher-S

porous gelatin microcarrier.

Size = 170 µm. Arrow

indicates live keratinocyte.

26

The CultiSpher-S microcarriers have a porous structure which greatly enhances

the surface area for cell adhesion with pores that range in size from 10-20 µm. The

adhesion properties of the microcarriers makes them suitable for cell expansion for

transplantation purposes or large scale biomolecule production (169) and several

cell types have been successfully cultured on CultiSpher-S (170). The porosity of

the microcarriers also contributes to the mass transfer properties of the scaffold by

creating an open scaffold where nutrients and oxygen can passage freely.

Previous work utilising the CultiSpher-S microcarriers reflect the versatility of

possible applications for porous gelatin carriers. The work includes investigations

of microcarrier culture of primary keratinocytes aimed for transplantation and the

transplantation of keratinocytes to full thickness wounds in rats (171, 172), the

microcarriers as soft tissue guiding scaffold in mice (173) and humans (174), and

engineering of bone and cartilage-like tissues with the aid of gelatin microcarriers

(175, 176). The suitability of gelatin microcarriers for keratinocyte expansion and

as transplantation vehicle has been established, accordingly in paper I the aim was

to investigate the gelatin microcarriers for their scaffolding properties for

epidermal cells without added cells. The foremost questions were which cells in

the wounds would populate the scaffold and what organisation the tissue would

obtain. Additionally, the use of CultiSpher-S in suspension in in vitro wounds was

tested. A suspended three dimensional matrix that is malleable extends potential

to use in different types and forms of skin lesions where no pre-fabrication of a

scaffold would be needed.

Modelling skin and wound healing

There is no fully functional in vitro modelling system to model the complexity of

the wound healing process of human skin. Basic molecular understanding of

wound healing has primarily been discovered by the use of animal models. This

hampers the transition from pre-clinical studies to clinical studies (177). The few

27

clinical studies in turn are difficult to implement due to the complex patient group

and differences in underlying conditions that can affect the wound healing process.

Animal models can never fully represent human wound healing and the use of

animal models should always be restricted from an ethical point of view and (178).

Modelling systems for investigating any physiological process or pathological

responses are still of undisputed importance to identify biological targets and

treatment strategies. For wound healing, numerous factors dictate the choice of

model. General healing or only limited parts of the process can be of interest, or

delayed or excessive healing, which all require different models to investigate

(179).

The simplest of models to investigate wound healing responses is the use of single

cell layers of keratinocytes in a scratch assay. This is merely a method to

investigate keratinocyte proliferation and migration, and it has been used for

investigating migration of keratinocytes in papers II and III. Using single cell

layers and scratch assays limits the conclusions that can be drawn from the assay

to concerning keratinocyte activation and migration only (180, 181). Studies on

the contractile properties of fibroblasts or fibroblasts extracted at different stages

of scar maturation are also performed with the aid of simpler in vitro models, often

accompanied by contraction assays involving collagen gels (182). However, the

complexity of wound healing with paracrine and autocrine interplay between the

dermis and the epidermis cannot fully be reflected in cell assays. To add levels of

complexity, skin substitutes have been developed and used for wound healing

assays alongside wound treatment (183, 184). The organotypic models where

individual components can be manipulated are an important tool for identifying

specific factors or mechanisms of action, but remain incomplete in barrier function

and ECM composition.

28

The benefit of using a whole animal model is the inclusion of circulatory and

systemic responses. Model animals frequently used in pre-clinical studies of

wound healing are rats and mice. Transgenic mouse models have contributed to

the field in terms of specific gene products and their contribution to the wound

healing process (185, 186) and by providing disease models (187, 188). The

translational problem that stems from using rodents is that the biology of the skin

and subsequently the wound healing process in rodents is different from human.

The skin of rats and mice has fur and contains a subcutaneous layer of striated

muscle, the panniculus carnosus. The overlaying skin is loosely attached to the

supporting structure (189) and a combination of loose skin and its contractile

properties makes wound contraction the main mode of action of healing in rodents

(190). Excisional wounds in rodents require splinting to allow investigations on

re-epithelialisation and granulation tissue effectively, which introduces an external

impact on the wound healing process (191).

In terms of structure and physiology of the skin the ideal animal model for studying

wound healing is the porcine model (192, 193). The wound healing process is in

general similar in human and pig, and pig skin has a similar vasculature and the

same proportions of epidermis and dermis as human skin. The physical size of the

animal enables multiple wounds to be placed on one animal (179). Despite the

benefits of employing pigs as model animals their use is restricted due to high costs

and need for specialised facilities (194).

Experimental treatment strategies for wound healing in human subjects are

performed at opportune conditions in clinical settings on chronic wound patients

and on split-thickness donor sites (195). The infliction of incisional wounds have

long been implemented (196) though the use of human subjects is always ethically

constrained due to the pain and discomfort that experiments may cause, and the

possibility of obtaining persisting scars. The use of human wound tissue samples

29

in combination with large animal models has been suggested (193). In a consensus

statement, Baird et al. propose hypothesis construction to be based on human

tissue samples and analysis by genomic and proteomic methods to identify targets

and mechanisms that later would be investigated in animal models, and that tissue

validation studies could contribute to more translationally relevant studies.

In research concerning the barrier function of the skin, human subjects have been

exposed to known irritants by the means of occluded patch testing. The reaction in

the skin has traditionally been evaluated on the basis of inflammatory symptoms

(197). The use of human subjects is limited by the discomfort caused and

subsequently that the exposure times have to be kept short. The clinical

manifestations of irritants are difficult to determine visually, and one of the

strongest arguments against patch testing has been the subjectivity of the analysis

(198). The same concerns are raised in the use of the classic Draize test for

toxicology and irritant testing. The Draize test is performed on small rodent eyes

or skin and the adverse effects are recorded. Deemed cruel, the method has been

challenged due to the inherent differences between humans and the model species

(199). Alternative methods based on human reconstituted skin models are being

developed, out of which few have reached the last validation steps according to

the guidelines set by ECVAM, the European Centre for Validation of Alternative

Methods. These lack the full barrier system of the skin which is of utmost

importance for predictive results in irritant testing.

Human full thickness skin in vitro

The uniting factor in the four papers that constitute this thesis is the use of human

full thickness skin in vitro. In papers I, II and III the human full thickness wound

healing model has been used. In 1998, Kratz introduced a model based on tissue

culture of viable human full thickness skin (200). Skin biopsies were cultured

submerged in culture medium for up to four weeks, during which time cells

30

remained viable in the tissue. Standardised full thickness wounds are created in the

tissue with the aid of biopsy punches. The full thickness wounds are prepared with

a punch that is three or four millimetre in diameter. A biopsy is taken with a larger,

six or eight millimetre punch, creating single biopsies with circular wounds in the

centre. Wound edge keratinocytes are activated and migration takes place as the

keratinocytes re-epithelialise the three to four millimetre wounds in an average of

seven days (Figure 6 A). This takes place when wounds are cultured in a

maintenance medium consisting of Dulbecco’s modified Eagle medium (DMEM)

supplemented with 10 % foetal calf serum (FCS). The cultivation of wounds in 2

% FCS results in a viable cell population but no re-epithelialisation. The 2 % FCS

culture condition could be considered as a non-healing environment with depletion

of nutrients, and can be utilised to investigate treatment interventions for chronic

wounds.

The progress of re-epithelialisation is monitored by tissue preparation and paraffin

sectioning, or cryosectioning, followed by routine haematoxylin and eosin (H&E)

staining (Figure 6). The re-epithelialisation can be measured or scored as complete

or incomplete. In the occasion of a hair follicle present in the wound bed, the

wound needs to be excluded from the analysis if only wound edge keratinocyte

Figure 6. (A) Haematoxylin and eosin staining of a fully re-epithelialised in vitro wound after seven days of culture in Dulbecco’s modified Eagle medium with 10

% foetal calf serum. (B) Representative image of wound edge of an in vitro wound presenting no re-epithelialisation. Scale bar = 500 µm.

31

contribution is of interest. Since epidermal cells are present in the sheet of the hair

follicle the origin of the cells in the neoepidermal tongue cannot be determined.

The tissue sections can further be investigated with histological or

immunohistochemical methodology.

In paper IV, human full thickness skin was used to evaluate its suitability to test

cytotoxic effects of irritants. To test cytotoxicity the skin was left intact as this

would be the case in a normal exposure of human skin to harmful compounds. The

benefit of using full thickness human skin is the intact barrier system that the

epidermis and the dermis forms. Problems arising from using reconstituted skin

models or skin cell assays are the vulnerability of these systems and the over-

prediction this brings. The localisation on the body from which a sample for

experiments is extracted plays an important role for irritant testing: thinner skin

will be over-predictive for a reaction on a body localisation with thicker skin. This

should be considered when planning for experiments. As selecting localisation of

tissue sample is important for irritant testing, the same principles can be applied to

inclusion in re-epithelialisation experiments. The foremost inclusion/exclusion

criteria of interest would be any underlying condition that severely affects wound

healing, like diabetes or other venous insufficiencies.

Skin, wound healing and reciprocity

Cutaneous wound healing is a dynamic process where the different phases overlap

in time, and involve all constituents of the wound healing environment from

resident cells to small fragments of the ECM. The reciprocity of the interactions

between cells and the ECM has long been established and wounding is an example

of the adaptability of the reciprocal interactions that take place when extensive

disturbance to tissue homeostasis occurs. The term dynamic reciprocity was coined

to describe how the interactions take place bi-directionally and change in response

to cues from the microenvironment (201). The components of the ECM take part

32

in all steps of the wound healing process and not only as a temporary matrix (202).

The first response to wounding, the haemostatic response, is a demonstration of

direct interaction between components of the ECM and the initiating factors in the

wound healing process. As wounding occurs, cells and platelets in the vasculature

are directly exposed to ECM components and haemostasis is initiated (203).

Another example of direct contact is the extravasation of neutrophils and

monocytes, where binding of cells to the affected endothelial cells and the exposed

ECM is crucial for extravasation to occur. Once in the damaged tissue, monocytes

bind to fibronectin in the wound environment which initiates phagocytic responses

(204) and differentiation into macrophages (205).

The ECM provides not only the structural integrity of the tissue but also the cellular

context for the cells present in the skin (206). Cells require to be supported and

connected to neighbouring cells or an ECM in order to function. The ECM proteins

support functions of cells by the multiple modes of interaction they present, and

the cells respond to changes in the ECM (207). Integrins are the key players in

conveying biochemical and structural changes from the ECM to cells, and

integrins possess inside-out and outside-in signalling capacity. By binding the

RGD motif, integrins are involved in the crosstalk between ECM proteins and the

actin cytoskeletons (208), cell adhesion and migration (209), as well as growth

factor responses (210). The ECM itself is a repository for growth factors that can

be locally released to influence cells in the surrounding as a rapid response when

wounding occurs (211). Several ECM proteins present affinity for both cell

adhesion and growth factors which in turn-fine tunes the local availability of a

growth factor to the vicinity of cell surface receptors (212). As important as a fast

response to disturbed homeostasis is, is the modulation of the ECM when cellular

functions need to be downregulated at the remodelling phase to avoid excessive

scarring and damage to the tissue (211, 213).

33

Fibroblasts are responsible for the deposition of granulation tissue and the ECM

during wound healing. From being quiescent cells in the dermis, fibroblasts are

activated to migratory cells with biosynthetic tasks (214). Mechanical stimulation

through cues from the ECM (215) and activation by TGF-β (216) signals resident

fibroblasts to differentiate into contractile myofibroblasts. Keratinocyte-derived

interactions have also been suggested, since fibroblast in co-culture with

keratinocytes acquire contractile phenotypes after a short period of time (217). The

interplay between keratinocytes and fibroblasts are prominent in the late

inflammatory and the proliferative stages of the wound healing process (218). The

importance of ECM and fibroblast support for keratinocyte proliferation are

known and primary keratinocytes have long been cultured on feeder layers of

irradiated fibroblasts. Demonstrated in the 1970s (219) the effects have been

shown to be reciprocal: the keratinocytes influence the fibroblasts to express

keratinocyte growth factor (KGF), IL-6 and GM-CSF to sustain a proliferative

environment (220, 221). KGF expression in fibroblasts can also be induced in vitro

by IL-1, TNF-α, PDGF and serum (222), factors that all are initiating factors in the

cutaneous wound healing response. Recent work by Larjava et al. (223) showed

keratinocytes to directly affect fibroblast function via secreted vesicles.

Keratinocytes contain microvesicles that can be shed, and when stimulating

fibroblasts with microvesicles, pro-granulation tissue genes were upregulated in

the fibroblasts in a dose-dependent manner. A third of the regulated genes were

involved in TGF-β signalling. Furthermore, microvesicles were shown to regulate

release of MMPs, IL-6 and IL-8. The role of microvesicles in the wound healing

context is still to be elucidated, but it is one example of the various ways of

interplay between the two main compartments of the skin and the cells that reside

therein.

The complex nature of the wound healing process and its dynamic reciprocity

speaks for modelling systems that take into account the contextual aspect of

34

individual steps in the process. The use of human full thickness skin is a means of

investigating the wound healing process in vitro without use of whole animals, yet

still taking into account the epidermal and dermal contributions to re-

epithelialisation. The dermis and its ECM supports the function of epidermal

keratinocytes both under normal and wound healing conditions. By utilising

human full thickness skin in vitro with all its structural components, a

comprehensive look on re-epithelialisation and barrier function of the skin can be

achieved.

35

AIMS OF THESIS

The overall aim of the work presented in this thesis was to investigate viable

human full thickness skin in vitro to enable investigations on wound re-

epithelialisation and irritant responses.

The specific aims of the included papers:

I. To investigate the effect of providing resident cells in in vitro wounds with a

porous gelatin microcarrier scaffold in suspension.

II. To develop a method to investigate the fate of keratinocytes and melanocytes in

the re-epithelialisation process when transplanted to human in vitro wounds in

suspension or on gelatin microcarriers.

III. To investigate the effects of acidic pH on re-epithelialisation of human in vitro

wounds and keratinocyte function to evaluate the efficiency of lowering pH of the

wound environment as adjuvant therapy for wound healing.

IV. To investigate human full thickness skin in vitro as a modelling system for

cytotoxic effects of irritant compounds topically applied to human skin.

36

37

MATERIAL AND METHODS

Primary cell culture and media

Keratinocytes and melanocytes were cultured in their respective maintenance

medium (Table I) with medium changes three times per week. Fibroblast medium

(FM) consisting of Dulbecco’s Eagles modified medium (DMEM) supplemented

with 10 % foetal calf serum (FCS) was used for washing tissue and cells during

cell preparation, and for centrifuging cells after passaging with trypsin. Primary

keratinocytes and melanocytes (Figure 7) were prepared from full thickness skin

biopsies under sterile conditions. Subcutaneous fat was removed by sharp

dissection and the skin was washed in sterile phosphate buffered saline (PBS). For

keratinocyte preparation, skin was cut into strips measuring one centimetre wide

and three centimetres long and submerged in a DMEM solution containing 25

U/mL dispase at 4 C overnight. The epidermis was lifted off with forceps, minced

with scissors, and placed in trypsin and ethylenediamine tetra acetic acid (EDTA)

(0.25 % and 0.02 %, Gibco, Thermo Fisher Scientific, Waltham, MA) solution for

15 minutes at 37 C. The solution was agitated every second minute by vortexing.

The trypsin was inactivated with centrifugation at 300 G in FM. The resuspended

epidermis-cell suspension was cultured in keratinocyte serum free medium

(KSFM), and non-adherent cells and debris was removed by rinsing with KSFM

two days after seeding. Melanocyte preparation required removal of subcutaneous

fat. The remaining epidermal and dermal tissue was minced with scissors, and

placed in trypsin-EDTA solution and agitated with a magnetic stirrer continuously

for 40 minutes at 37 C. After agitation, the tissue was left to sediment and the

supernatant was collected, centrifuged in FM and resuspended in melanocyte

growth medium (MGM). The procedure was repeated three to four times, and the

supernatants were collected for seeding in appropriate culture vessels, depending

38

on the amount of starting material. Culture medium change took place the day after

seeding.

Table I. Composition of cell culture medium

Medium Abbreviation Basal medium

Supplements Manufacturer

Keratinocyte serum free medium

KSFM KSFM with L-glutamine

25 µg/mL bovine pituitary extract (BPE) 1 ng/mL epidermal growth factor (EGF) 50 U/mL penicillin 50 µg/mL streptomycin

Gibco, Thermo Fisher Scientific

Melanocyte growth medium

MGM PC-1 base medium

2 % PC-1 supplement 1 % L-glutamine 5 ng/mL basic fibroblast growth factor (bFGF) 24.6 g/mL N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate (dbcAMP) 50 U/mL penicillin 50 µg/mL streptomycin

Base: Lonza Supplements: Sigma-Aldrich

Fibroblast medium

FM

Dulbecco’s

Modified Eagle Medium

10 % foetal calf serum 50 U/mL penicillin 50 µg/mL streptomycin

Gibco, Thermo Fisher Scientific

Figure 7. (A) Colony of primary human keratinocytes. (B) Primary human melanocytes. Scale bars = 1000 µm.

39

Microcarriers and spinner flask culture Porous gelatin microcarriers from PerCell Biolytica (Åstorp, Sweden) were

applied to in vitro wounds in papers I and II. The aim of paper I was to investigate

the scaffolding effects of the microcarriers. The CultiSpher-S microcarriers are

based on highly crosslinked porcine gelatin type A and fully biodegradable (Figure

5). Cell culture on microcarriers requires

agitation of the microcarriers suspended in

culture medium which is done by using spinner

flasks for culture (Figure 8) (Techne,

Staffordshire, UK). The spinner flask is a basic

bioreactor and one of the most commonly used

in cell culture applications (224). Bioreactor

design spans from microscale perfusion

chambers to industrial size rotating wall

vessels (225). The culture of cells on

microcarriers in spinner flasks can be

performed when the goal is to upscale the

culture of adherent cells (226), since the use of

spinner flasks reduces medium consumption

and hence the costs. The spinner culture also improves oxygenation and the

distribution of nutrients for the cultured cells (224). For maximal cell proliferation

to be achieved, mass transfer needs to be reached without the negative effects of

the hydrodynamic forces present in the spinner culture (226). The cell damage in

spinner flasks mainly rise from collisions between the microcarriers or with the

agitator, or with interactions with liquid eddies when the cell-microcarrier

suspension is agitated (227). An agitation rate enough to suspend the microcarriers

can be sufficient for adequate oxygenation. However, if the spinning is the only

mode of aeration of the culture the agitation speed might have to be increased. A

Figure 8. Spinner flask culture set-up with spinner flask and stirrer placed in incubator.

40

liquid area can absorb only a limited amount of oxygen and this has to be

considered when the culture volume of the spinner culture is decided: a larger

volume of liquid will decrease the liquid surface/volume ratio and result in

decreased oxygenation and lower cell yield (226). Other considerations than

agitation speed and culture volume that need to be addressed are the carrier and

cell type to make sure the adhesion of the cells is adequate. The possible reactions

of the cells to the forces in culture should also be considered and the integrity of

the microcarriers themselves exposed to the planned agitation speeds and culture

times should be monitored (224).

The property of the microcarriers as tools for cell transplantation was utilised in

paper II where cells were expanded on microcarriers before transferred to in vitro

wounds. Keratinocytes and melanocytes were mixed with microcarriers at a

concentration of 50 000 cells/mg microcarriers. The suspension was added to

spinner flasks and agitated at 35 RPM for five minutes every hour for the first 24

hours of culture to allow cell attachment. After the initial phase, the culture was

agitated continuously and half of the culture medium volume was changed three

times per week. When microcarriers were transplanted to in vitro wounds, the

spinner flask was manually agitated to obtain a uniform solution to remove the

microcarrier samples from.

Viability assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

assay is a colourimetric assay for assessing metabolic activity of cells. The

assay is based on the activity of mitochondrial reductases, which can reflect

the number of viable cells in a sample. The enzymes reduce the yellow MTT

salt to purple insoluble formazan. Once dissolved in dimethyl sulfoxide

(DMSO) or isopropanol, the produced formazan is measured

41

spectrophotometrically at 570 nm to quantify the absorbance (228). In paper

II, MTT assays were performed on keratinocytes and melanocytes in

adherent culture, and on both cell types cultured on microcarriers in spinner

flask culture. In paper III, keratinocyte viability was investigated after

exposure to acidic pH. Cultures were incubated in MTT (Sigma-Aldrich, St.

Louis, MO) dissolved in DMEM 0.3 mg/mL for four hours in the dark, after

which incubation with DMSO took place for 10 minutes. In paper IV, the

MTT assay was employed to investigate the cytotoxic effect of irritant

compounds applied to human full thickness skin. Skin biopsies were

submerged in MTT solution for four hours and the formed formazan was

dissolved in isopropanol overnight at room temperature.

Migration assay

Migration of carboxyfluorescein hydroxysuccinimidyl ester (CFSE) stained

keratinocytes and melanocytes (paper II) and keratinocytes exposed to acidic pH

(paper III) was investigated by performing a scratch assay (180). Cells were seeded

and cultured overnight to a confluent layer. Prior to the migration assay, the

cultures were incubated with mitomycin C (Roche Diagnostics, Basel,

Switzerland) for two hours at 37 °C. Mitomycin is a DNA cross linker that will

inhibit proliferation in the culture and therefore excludes the contribution of

proliferation to re-population of the scratch (229). The cell layer was then

scratched with a p200 pipet tip to obtain a cell free area, which was monitored over

a series of hours to investigate the migration of the cells into the cell free area

(Figure 9). Analysis of the migration assay includes tracing the remaining areas

that are not re-colonised by cells in the image processing analysis software FIJI

(230) and compare the results with time point zero hours. Reults are presented as

coverage of the initial cell free area in percent.

42

Quantitative real-time polymerase chain reaction In paper III, the relative mRNA expression of certain genes of interest was

investigated in keratinocytes exposed to pH 5.0 and pH 6.0 compared with

untreated controls with the aid of quantitative real-time polymerase chain reaction

(qRT-PCR) (231). Gene expression assays and reagents were purchased from

Applied Biosystems (Foster City, CA). Total RNA was isolated from cells with

High Pure Isolation Kit (Roche Diagnostics) and quantity and quality was

measured with a NanoDrop 2000 (Thermo Fisher Scientific). RNA was transcribed

to cDNA with High Capacity cDNA Reverse Transcription Kit (Applied

Biosystems) and PCR was run using TaqMan Fast 96-well plates in a HT7900

thermocycler (Applied Biosystems).

The use of Taq polymerase for DNA replication applications was a break through

when introduced. Previously polymerase activity was restricted by the high

temperatures required to denature the newly formed DNA strands to enable the

single strands to act as templates for the next round of amplification. The Taq

polymerase was discovered in the bacterium Thermus aquaticus and isolated by

Chien et al. in 1976 (232). Thermus aquaticus is resident in hot springs so the

polymerase could now withstand the temperatures up to 95 °C that are required for

Figure 9. Scratch assay and progression of migration. Time point zero hours (left) with a distinct scratch in the confluent keratinocyte layer compared with time point 24 hours (right) of the same well where keratinocytes have re-populated the scratched area. Scale bar = 500 µm.

43

a PCR, and enabled reaction in a single tube and no addition of polymerase in the

middle of running the PCR (233). The primer assays used in paper III were

TaqMan probe based gene expression assays. TaqMan probes are primer probes

that were developed by Roche Molecular Diagnostics and Applied Biosystems

enabling real time monitoring of the accumulation of PCR product during the

reaction. The probes are designed with a fluorophore at the 5’ end and a quencher

at the 3’ end which quenches the fluorescent signal when in close proximity of the

reporter fluorochrome, i.e. when the probe is intact. As the polymerisation

proceeds, the exonuclease activity of the Taq polymerase cleaves the probe on the

target sequence and allows a fluorescence signal to be detected. This enables

continuous monitoring of the amplification of target sequences during the

exponential cycles of the PCR and the relative quantification of the expression of

genes of interest.

To analyse the data from gene expression experiments in paper III, a method to

establish relative expression termed the 2-ΔΔCt method was used (234). The Ct value

(cycle threshold value) of the gene of interest is the number of amplification cycles

required to reach a threshold of fluorescence signal above the background and

baseline signals. The basic assumption made is that one cycle difference in

reaching the threshold represents a doubling of starting template in the PCR. The

difference in Ct value, and thus the fold change, is obtained by relating the Ct value

of the target gene to an endogenous control (hydroxymethylbilane synthase

(HMBS) in paper III) and to an internal calibrator, in our case the untreated control

cells. The application of endogenous controls normalises the expression and

controls for variations in assays and experiments, as well as cDNA yield.

Human in vitro wound healing model

The in vitro wound healing model utilised in papers I, II and III is based on the

culture of viable human full thickness skin. Preparation of in vitro wounds requires

44

full thickness skin, cleared from subcutaneous fat. Skin samples were washed and

kept moist in PBS. The adipose tissue was removed with scissors. Full thickness

wounds were prepared by using standard 3 mm biopsy punches (Figure 10 A and

B) to create circular wounds. This resulted in standardised full thickness wounds

spanning over the dermo-epidermal junction into the dermis. The wounds were

separated from the larger skin specimen with a 6 mm biopsy punch, to create 6 mm

biopsies with a central 3 mm wound (Figure 10 C). The wounds were cultured in

individual wells in 24-well plates in FM with medium changes three times per

week in all three projects (Figure 10 D). In paper I, wounds were incubated in cell

culture inserts (Figure 10 E). The placement of the wound in the air-liquid interface

induces stratification of the epidermis, with cornified keratinocytes in the

superficial layers of the newly formed epidermis of a re-epithelialised wound.

Wounds with hair follicles present were excluded from analysis.

Non-occlusive topical exposure

In paper IV, full thickness skin in vitro was used to model reactions in the skin

after exposure to irritant compounds. Cut-off strip tubes in pairs or in sets of three

were used to create confined areas of exposure on the skin (Figure 11). The rings

were pressed into skin that was cleared from adipose tissue to create chambers with

exposure areas of 0.80 cm2. Sodium dodecyl sulphate (SDS) and other test

substances, as well as PBS as DMEM controls were applied to the formed

chambers and the samples were placed in an incubator. After pre-defined exposure

times the centres of the exposed areas were extracted with a 3 mm biopsy punch.

Biopsies were washed in PBS and submerged in 0.3 mg/mL MTT-solution (in

DMEM) for four hours at 37 C. As described earlier, the formed formazan was

precipitated using isopropanol at room temperature overnight. The optical density

was measured using a plate reader (Versamax, Molecular Devices). Results were

presented as relative to control substance values and expressed in percent.

45

Paraffin embedding and sectioning

Paraffin embedding of tissue samples and sectioning was performed in paper III.

Paraffin embedding is routine methodology to obtain tissue samples of high

morphological standard that can be stored for long periods of time. To begin the

embedding process the in vitro wounds were fixated with 4 % formaldehyde (PFA)

(HistoLab, Gothenburg, Sweden) for four hours. The fixation substance and length

of fixation depends on the tissue sample and downstream application.

Formaldehyde in aqueous solution forms methylene glycol which reacts with

several side chains of proteins. The formed reactive side groups react with each

Figure 11. (A) Cut-off strip tubes in sets of three for creating restricted exposure

areas on human skin for cytotoxicity testing. (B) Tubes pressed onto viable full

thickness skin for non-occluded exposure to irritant.

Figure 10. (A) Standard three and

six millimetre biopsy punches for

creation of in vitro wounds. (B)

Full thickness skin sample with

two in vitro wounds created with a

three millimetre biopsy punch. (C)

Single in vitro wound extracted

from the whole skin sample with a

six millimetre biopsy punch. (D)

Submerged in vitro wound. (E) In

vitro wound cultured in cell culture

insert.

46

other or combine with hydrogen groups, thereby causing fixation of the proteins

(235). After washing in PBS, tissue was dehydrated in ethanol. Lastly, the samples

were submerged in Tissue clear, which is miscible with melted paraffin. After

soaking in paraffin overnight, the samples were embedded in paraffin and left to

cool to obtain hard tissue samples that can be sectioned, and stored at room

temperature. Tissue was sectioned using a Leica RM2255 microtome (Leica,

Wetzlar, Germany) in 10 µm thick sections and baked on glass slides at 56 °C

overnight.

Cryosectioning

Cryosectioning of tissue biopsies in the size range of the in vitro wounds is a faster

method to obtain tissue sections compared to paraffin embedding and sectioning.

However, the process of cryosectioning may lead to loss of structural morphology

compared with fixated paraffin samples. Cryosectioning was preferred in papers I

and II due to the presence of microcarriers in the wounds: early experiments

revealed loss of microcarriers in the dehydration process and cryosectioning,

which requires less preparation and change of solutes, was chosen as sectioning

method. Samples were attached to sample holders with optimal cutting temperature

compound (OCT) (HistoLab) and snap frozen by submerging in liquid nitrogen.

Sectioning was performed with a Leica CM3050 cryostat at -28 C and tissue

sections were placed on Superfrost plus glass slides (Thermo Fisher Scientific).

The thickness of prepared sections was 10 µm for routine staining described below

and 50 µm for confocal imaging in paper I.

Haematoxylin and eosin staining

Tissue staining with haematoxylin and eosin (H&E) was used in papers I, II and

III to observe morphology and to investigate the degree of re-epithelialisation of

the wounds. H&E is the most widely used staining method for tissue morphology

investigations and histopathological assessments. Paraffin embedded sections in

47

paper III were deparaffinised in tissue clear, rehydrated in ethanol, and

subsequently washed in de-ionised water prior to staining. Frozen sections in

papers I and II were air dried and washed in PBS to remove the OCT. Sections

were stained with haematoxylin, washed under running tap water and stained with

eosin. After staining, sections were dehydrated in ethanol and mounted with

mounting medium (Mountex, HistoLab) and a cover slip. The times for staining in

haematoxylin and eosin solutions varied depending on what type of fixation and

preservation was used, with generally shorter staining times for frozen sections.

Eosins are routinely used as counterstains to haematoxylin nuclear staining. They

are acidic and stain basic positively charged structures, and by staining with eosin

one can distinguish cytoplasms of different cell types and different types of

connective tissue in varying shades of red and pink. The intense staining of red

blood cells can also facilitate the localisation of small vessels in the dermis of the

skin. The differentiation of the stain takes place when the slides are dehydrated in

alcohol after staining and is essential to obtain information and comprehensive

images of the tissue slide. The haematoxylin stains are extracted from the logwood

and are not stains in themselves: haematein naturally formed by oxidation carries

the colour properties. Additionally, a mordant is required to increase the affinity

of haematein to tissue. Mayer's haematoxylin used in papers I, II and III is an alum

haematoxylin where the added mordant is aluminium salt. The metal cation gives

a net positive charge to the complex of dye and mordant, and binding of anionic

structures such as nuclear chromatin is made possible. Haematoxylin stains nuclei

in a red stain that is blued by washing in a slightly alkaline solution, most often tap

water. This results in the dark blue colour that characterises the haematoxylin stain

(235). All sections were visualised using an Olympus BX41 light/fluorescence

microscope (Olympus, Solna, Sweden)

48

Immunohistochemistry

Immunohistochemical staining was utilised in papers I-III to visualise, localise and

identify different proteins. The basic principle of immunohistochemistry is the use

of targeted antibodies for a specific antigen of interest. The primary antibodies are

targeted with secondary antibodies conjugated with a fluorochrome or substrate

for enzymatic detection of the primary antibody. Fluorescent secondary antibodies

were used in all three projects (Alexa Fluor 488 and 546, Molecular Probes,

Thermo Fisher Scientific). In papers I and II, keratinocytes were targeted by

staining with antibodies against both acidic and basic cytokeratins. In paper I, this

was performed to visualise the neoepidermis of the wounds and to quantify the

thickness, and in paper II for distinguishing resident keratinocytes from the CFSE-

stained, transplanted keratinocytes. In paper I the markers of a mature epidermis

were targeted to investigate the maturation of the thick neoepidermis seen in the

wounds with administered microcarriers (Table II).

Cryosectioned tissue samples (paper I and II) were left to air dry for 30 minutes

prior to immunohistochemical staining. Paraffin embedded slides were treated

with Tissue clear and washed in 99.5 % ethanol and 95 % ethanol prior to staining.

All sections were fixated with 4 % PFA except sections intended for staining with

antibody against laminin 5. These were fixated with cold acetone as suggested by

manufacturer of the antibodies. After washing in PBS, unspecific binding sites for

primary antibodies were blocked with 2.5 % bovine serum albumin (BSA).

Primary antibodies were applied in a humidified chamber for 60 minutes for 10

µm sections, and at 4 °C overnight for 50 µm sections. Secondary Alexa-

conjugated antibodies were applied in a humidified chamber in the dark for 60

minutes. Slides were mounted with mounting medium Prolong Gold (Thermo

Fisher Scientific) containing 4’, 6-diamino-2-phenylindole (DAPI) for nuclear

staining. Control samples with omitted primary antibodies were employed to

ensure specificity of the secondary antibodies. The primary and secondary antisera

49

used in the studies have been used in previous studies and tested for specificity and

cross-reactivity (236-239).

Table II. Antigens targeted with immunohistochemical staining in paper I

Carboxyfluorescein hydroxysuccinimidyl ester (CFSE) staining

Cell staining with 5(6) carboxyfluorescein N-hydroxysuccinimidyl ester (CFSE)

is conventionally used for proliferation measurements of lymphocytes. The non-

fluorescent pro-dye passively moves over the cell membrane due to the presence

of two acetate groups (244). Once inside the cell, intracellular esterases remove

the acetate groups and a green fluorescence is yielded (Figure 12). The membrane

permeability of the molecule is reduced and the succimidyl groups present form

covalent bonds with amino groups, mainly lysine residues. The now covalently

linked conjugates of the stain can persist in lymphocytes over months and results

in a fluorescent staining that is sequentially halved at every cell division.

In paper II we have employed CFSE-staining for passive staining of primary

keratinocytes and melanocytes for tracing transplanted cells in tissue sections of in

vitro wounds. Keratinocytes and melanocytes were stained adherently according

to same protocol with caution to expose the stain solution or the stained cells to

bright light. CFSE (Molecular Probes, Thermo Fisher Scientific) was diluted in

DMSO to 5 mM, and diluted to working concentrations in pre-warmed (37 °C)

PBS. Three millilitres of CFSE solution was added to adherent cells in a 75 cm2

Antigen Reference

Keratin 5 Keratin expressed by keratinocytes in the basal layer

of epidermis, expressed concurrently with keratin 14 (240)

Keratin 10

Keratin expressed by keratinocytes in the superficial

layers of epidermis, expressed concurrently with

keratins 1 and 2

(241, 242)

Laminin, alpha

5

Extracellular anchoring filament and major

component of hemidesmosomes in basal membranes (46)

Ki-67 Nuclear antigen present only in proliferative cells (243)

50

flask and the flask was agitated for two minutes. Cells were incubated at 37 °C for

15 minutes and medium was changed to pre-warmed cell culture medium. After

30 minutes in the fresh medium cells can be harvested or left for expansion.

Keratinocytes and melanocytes cultured on microcarriers in paper II were stained

as described prior to attachment to the microcarriers.

Flow cytometry

Flow cytometry enables simultaneous measurement and analysis of several

characteristics of cells or particles using light scatter properties. Standard

applications are cell counting and cell sorting as well as analyses based on

Figure 12. Schematic description of the pro-dye of CFSE that can pass freely over

the cell membrane, and that after cleavage by intracellular esterases obtains the

fluorescent state and upon binding to intracellular proteins is retained in the

intracellular compartment. Adapted from Parish, 1999 258

51

fluorescently labelled markers of interest. Measurement of CFSE intensity with a

flow cytometer was performed to establish the proliferation of the CFSE stained

cells on the microcarriers and cells cultured adherently. Proliferation was

investigated after 72 hours in spinner flask culture. The CFSE stain is covalently

bound intracellularly and inherited at cell division as previously described. This

enables proliferation measurements under the assumption that the amount of CFSE

is halved at every cell division. By measuring the CFSE intensity of individual

cells with a flow cytometer, populations of cells that have equal amounts of CFSE

can be discerned. CFSE-stained keratinocytes and melanocytes cultured on

microcarriers were detached by incubation with trypsin for five minutes at 37 °C

followed by vortexing. Keratinocytes were passed through a 30 µm filter and

melanocytes through a 10 µm filter, washed and resuspended in PBS. A control

population for CFSE staining representing cells that have not divided was stained

as previously described six hours before flow cytometric analyses. Adherently

cultured CFSE-stained cells and six hour control cells were trypsinised, washed

and resuspended in PBS. Flow cytometric measurements were performed with a

Gallios flow cytometer (Beckman Coulter, Brea, CA) at 488 nm. Data was

analysed with Kaluza software (Beckman Coulter). Single cells were included in

the statistical analysis and cells overlapping the CFSE intensity of the six hour

control were excluded to obtain percentage of cells that had divided.

Fluorescence microscopy and confocal microscopy

In papers I and II fluorescence microscopy was used to localise antigens stained

with fluorescent secondary antibodies to identify proteins present in the re-

epithelialised wounds. The sections were visualised with an Olympus BX41

light/fluorescence microscope. Images were captured with a DP70 CCD camera

(Olympus) and overlay images were constructed in Adobe Photoshop CS5 (Adobe

Systems, San Jose, CA).

52

In paper I, the neoepidermis of wounds with microcarriers was investigated with

confocal imaging of the cytokeratin staining. Images were captured with a Zeiss

LSM 700 microscope with Zeiss ZEN software (Carl Zeiss AG, Oberkochen,

Germany) and images were constructed in FIJI (230).

Statistical analyses

Statistical analyses were performed in GraphPad Prism v. 5.0 (GraphPad, La Jolla,

CA). All graphs were constructed in GraphPad Prism v. 5.0. A probability level

of ≤ 0. 05 was considered significant.

Paper I. Neoepidermal thickness was measured digitally and wounds with

administered microcarriers were compared with control wounds without

microcarriers. Means were compared using Student’s t-test.

Paper II. A two-way analysis of variance (ANOVA) was performed to test

significance of viability of CFSE-stained keratinocytes and melanocytes compared

to non-stained control. A two-way ANOVA repeated measurements test was

performed followed by Bonferroni’s multiple comparisons test on the data from

scratch assays on migration of CFSE-stained keratinocytes and melanocytes. The

percentage of divided cells in microcarrier culture was compared with adherent

control cells and groups were compared with Student’s t-test.

Paper III. A Kolmogorov-Smirnov testing of the viability and migration data

revealed normal distribution and groups per time point were compared with

Student’s t-test in both cases. Fold change in the PCR of respective groups were

compared with controls cultured at normal pH with a Mann-Whitney U test.

Paper IV. Groups of the fifteen minute exposure experiments were compared with

Student’s t-test. One-way ANOVA was performed on experiments with more than

53

two groups. Non-linear regression analysis of cell viability and increasing

concentration of SDS was performed.

Ethical considerations

All cells used were of human origin. Primary cells were prepared from discarded

full thickness skin biopsies, rescued from routine reduction plasties or

abdomenoplastic surgeries. The rescue of tissue did not influence the proceeding

of the surgery in any way. All tissue samples were de-identified to ensure that

samples cannot be traced back to the patient to adhere to ethical guidelines

concerning the use of discarded human tissue (Law 2003:46).

54

55

SUMMARY OF PAPERS

The work presented in this thesis aimed at evaluating the use of human full

thickness skin in vitro. When cultured in a maintenance medium containing 10 %

FCS, the skin remains viable with cells that respond to wounding cues with

activation and re-epithelialisation. Moreover, intact full thickness skin exhibits a

complete barrier function with all components and constituting cells present. The

human in vitro wound healing model was used to study neoepidermal organisation

following administration of porous gelatin scaffolds. In this setting, a novel

fluorescent staining was evaluated for the tracing of transplanted cells, and to

evaluate the effects of modifying the wound healing environment. Finally, the use

of intact skin to study cytotoxicity responses was evaluated.

Paper I: Biodegradable gelatin microcarriers facilitate re-

epithelialisation of human cutaneous wounds – an in vitro study

in human skin

In previous studies published by our group, The CultiSpher-S gelatin porous

microcarriers have been used to investigate suitability as a dermal scaffold and as

a carrier for keratinocytes transplanted to rats (172, 174). In paper I, the human

wound healing model was used to test the effect of providing the resident cells in

the wounds with a porous, malleable gelatin scaffold. The results revealed

population of the scaffold after 21 days in culture. Wound edge keratinocytes

formed a thick neoepidermis (Figure 13) that stratified when cultured in the air-

liquid interface. The neoepidermis was analysed with regards to markers of mature

epidermis: keratin 5, keratin 10, and laminin 5 (Figure 14 A and D) using

immunohistochemical staining. Additionally, the expression of the proliferation

marker nuclear antigen Ki-67 was investigated. The cytokeratin expression was

found to be similar to the normal organisation of keratin 5 in the basal layers of the

epidermis and keratin 10 in the superficial layers. There was no increase in the

56

expression of the proliferation marker Ki-67 at the wound edges of the

experimental wounds compared with control wounds without carriers. This is

Figure 14. (A) Immunohistochemical staining against basal keratin 5 (green) and keratin 10 (red) of wound with incorporated CultiSpher-S microcarriers. (B) Immunohistochemical staining against keratin 5 (green) and keratin 10 (red) in normal unwounded skin. (C) Confocal image of microcarrier populated by keratinocytes, staining against pancytokeratin (red). (D) Immunohistochemical staining against laminin 5 (green) in wound with incorporated microcarriers, (E) normal unwounded skin and (F) control neoepidermis. Nuclear staining (blue) with 4, 6-diamidino-2-phenylindole (DAPI). Scale bars = 100 µm.

Figure 13. (A) Neoepidermal thickness after 21 days in tissue culture. Control: 47.6 µm ± 3.1, with microcarriers: 128.0 µm ± 17.4 (mean ± SD). (B) Haematoxylin and eosin staining of in vitro wound with incorporated microcarrier scaffold presenting a thick neoepidermis. Scale bar = 500 µm.

57

indicative of normal proliferation rates, and that the neoepidermis is the result of

migration of wound edge keratinocytes. The neoepidermis in the wounds receiving

microcarriers displayed a disorganised cell orientation. However, the keratinocytes

within the scaffold expressed laminin 5 (Figure 14 D), an important anchoring

filament in the basement membrane structure. The integrin expression in

keratinocytes and the distribution in the epidermis contributes to the polarity of

keratinocytes (245) and the integrins are associated with laminin 5. The expression

of laminin 5 by keratinocytes that are incorporated into the lower parts of the

microcarrier scaffold indicates an obtained polarity in the neoepidermis.

The porosity of the CultiSpher-S microcarriers serves two main purposes: it

increases the surface area for cell attachment for adherent cells, and it allows the

diffusion of nutrients and oxygen throughout the entire scaffold. The effect of the

porosity of the scaffold biomaterial was investigated by culturing wounds with

added gelatin in solution. This revealed incomplete re-epithelialisation of

experimental wounds and points to the importance of the porous structure of

CultiSpher-S as a guiding cue for wound edge keratinocytes. Structure-wise the

microcarriers fulfil several of the demands placed on a biomaterial aimed for

regenerative purposes (246) where the ultimate goal is to use biomaterials that

closely resemble and mimic the niche of the cells that are to be stimulated to

regenerate a certain type of tissue. Gelatin bares high molecular resemblance to

collagen, the main component of dermis. Collagen type I and III form thin collagen

bundles in the papillary dermis. The lower and major part of the dermis, the

reticular layer, consists of thick bundles of collagen I (247). The in vitro wounds

span over the dermo-epidermal junction into the loosely packed papillary dermis.

When inspecting the interface between microcarriers and the dermal wound bed,

no gap or capsule formation was observed. Previous work by Huss et al. (174)

showed no adverse effects when CultiSpher-S microcarriers were injected into the

dermis of healthy volunteers. The microcarriers were populated with fibroblasts

58

after 14 days and the gelatin degraded by 12 weeks. The microcarriers show here

another important property for regenerative biomaterials: since the niche is

changing over time, which certainly is true for a wound healing environment, the

microcarriers can adapt to the environment yet provide an initial structural

stability. If possible, a biomaterial should also support neovascularisation. This is

true for the gelatin microcarriers and was shown in the study by Huss et al. where

the neodermis was vascularized after 12 and 26 weeks (174). In the present study,

we observed capillaries in the dermis in the vicinity of the microcarriers and no

gap formation between the scaffold and the dermis. Keratinocytes had populated

the complete carriers as seen by confocal imaging of microcarriers in the wounds

(Figure 14 C). Taken together, these studies indicate that CultiSpher-S show

biocompatibility and can function as a guiding scaffold for both epidermal and

dermal regeneration.

Conclusions and future outlook

Human in vitro wounds with administered microcarriers presented a thick

neoepidermis with incorporation of the gelatin microcarrier scaffold

Keratinocytes in the neoepidermis expressed keratins in a similar stratified

organisation to native epidermis

Keratinocytes within the microcarrier scaffold expressed basal membrane

marker laminin 5

The porosity of the microcarrier scaffold contributed to the organisation

of the neoepidermis

No gap formation took place in the interface between scaffold and the

dermal wound bed

The in vitro wounds are cultured submerged in cell culture medium. In clinical

practice acute and chronic wounds are treated under moist conditions, which has

59

been extensively demonstrated to improve healing outcome. Future human studies

to evaluate the beneficial properties of a porous microcarrier scaffold on wound

healing need be performed to obtain relevant information of the organisation and

barrier properties of the neoepidermis in an in vivo environment.

CultiSpher-S microcarriers exhibit many beneficial properties for future

applications: the carriers can be topically applied or injected, they are highly

biomimetic and can be loaded with cells or growth factors. The loading properties

strongly contribute to the possible development of a transplantable scaffold which

could mimic the natural niche of the involved cells and adapt to and stimulate the

wound healing process. The CultiSpher-S are malleable and require no pre-

fabrication steps before application to any shape of wound, and are according to

our findings suitable as a tissue guiding scaffold for both epidermis and dermis.

Paper II: Tracing transplanted human keratinocytes and

melanocytes with carboxyfluorescein hydroxysuccinimidyl

ester (CFSE) staining

The human in vitro full thickness skin wound healing model was utilised in paper

I to investigate the potential of porous gelatin microcarriers as scaffolding for

resident cells. The culture of adherent cells in this type of microcarrier culture is

conventionally aimed to expand cells for subsequent transplantation, and the

porous structure responsible for the scaffolding effects is optimised for a large

adhesion surface and therefore a large number of cells. To use the wound healing

model to further investigate in vitro wound healing requires a means for tracing

transplanted cells in culture. The aim of paper II was to investigate the use of

carboxyfluorescein hydroxysuccinimidyl ester (CFSE) as a tracing staining for

primary keratinocytes and melanocytes. Clinically, keratinocytes are transplanted

when treating burns, and melanocytes when treating hypopigmentation conditions

60

like vitiligo. The use of CFSE-staining has traditionally been proliferation

monitoring in lymphocytes (248) and investigated with flow cytometry. We

investigated viability, migration, and proliferation of CFSE-stained keratinocytes

and melanocytes. We found CFSE- staining to not affect viability of either cell

type. Viability of keratinocytes at the seven day time point with 5 µM staining was

100.0 % ± 8.2 relative to unstained control 100.0 % ± 7.4. Viability of melanocytes

at the same time point and concentration was 114.0 % ± 19.0 relative to control

99.8 % ± 17.3. CFSE has been shown to be potentially toxic and modulating

surface protein conformation, which needs to be considered if surface proteins are

the target of the investigations (249, 244) however, for primary keratinocytes and

melanocytes the lowest staining concentration proposed by manufacturer was

shown to be sufficient and safe. Migration was investigated with scratch assays

and migration of both cell types was unaffected seen by a full repopulation of the

scratched area within 24 hours. Proliferation of CFSE-stained keratinocytes and

melanocytes was investigated with flow cytometric measurement, thus employing

the CFSE-staining for its conventional purpose. Retained proliferation is a

prerequisite for the microcarriers to be utilised clinically for transplantation since

a large number of cells is required for these procedures. Proliferation rates were

retained when the two cell types were cultured on microcarriers (70.4 % ± 37.9

divided cells in keratinocyte microcarrier culture compared with adherent control

88.8 % ± 5.4, and 26.3 % ± 21.0 compared with adherent control 20.46 % ± 17.2

for melanocytes). Both keratinocytes and melanocytes showed great variability

when cultured on microcarriers, with keratinocyte proliferation differing from one

spinner flask culture to the other. Technical shortcomings could be the reason for

the observed variability in keratinocyte cultures: the detachment of the CFSE-

stained cells from microcarriers with trypsin was more problematic to perform on

keratinocytes than on melanocytes. This corresponds to the level of adhesion the

two cell types present in adherent culture on cell culture polystyrene, where

61

melanocytes are easily detachable requiring substantially less time for

trypsinisation than keratinocytes. Filtration necessary for the subsequent flow

cytometric measurements could also have played part in the problem of obtaining

sufficient amounts of cells, and if keratinocytes were still attached to fragments of

gelatin these cells would have been lost before analysis.

The CFSE-staining withstood cryosectioning procedures. Green fluorescent

transplanted keratinocytes and melanocytes could be localised in tissue sections

after seven, 14 and 21 days of tissue culture (Figure 15). Keratinocytes were

Figure 15. (A) Re-epithelialised wound after seven days of culture with carboxyfluorescein hydroxysuccinimidyl ester (CFSE) stained keratinocytes transplanted in suspension indicated by arrow heads. Dashed lines indicate wound edges. Scale bar = 500 µm. (B) In vitro wound at day seven of culture with transplanted CFSE-stained keratinocytes and CFSE-stained melanocytes on microcarrier scaffolds. Section stained with antibody against cytokeratin to distinguish native keratinocytes (red), transplanted double-stained keratinocytes (yellow, arrows) and transplanted melanocytes (green, arrow head). Scale bar = 200 µm. Nuclear staining (blue) with 4, 6-diamino-2-phenylindole (DAPI).

62

transplanted in suspension as well as on microcarrier scaffolds. The wounds

receiving no microcarriers but CFSE-stained cells showed expected re-

epithelialisation times and incorporation of transplanted keratinocytes in the re-

epithelialised wound (Figure 15 A), thus no negative effect of the staining on re-

epithelialisation could be observed. Co-transplantation of CFSE-stained

keratinocytes and melanocytes was performed with the aid of microcarriers. Here

the two transplanted cell types were distinguished from one another by

immunohistochemical staining against cytokeratin and red fluorescent secondary

antibody. The double stained keratinocytes could be seen in orange/yellow

compared with the red stained native epidermis (Figure 15 B).

The application of CFSE-staining for tracing human primary keratinocytes and

melanocytes was shown to be suitable and easily applicable. The staining is readily

available, easy to use and requires no custom preparation of the cells. In the present

study CFSE-staining was used for tracing human primary keratinocytes and

melanocytes in vitro. CFSE-stained cells have been traced in vivo in mouse (250,

251), rat (252), and pig models (253) but further studies on toxicity of CFSE for

humans need to be performed before in vivo applications can be considered.

Conclusions and future outlook

Staining of human primary keratinocytes and melanocytes with 5 µM

CFSE did not significantly affect viability, migration or proliferation of

the cells

Administration of CFSE-stained cells to human in vitro wounds did not

affect re-epithelialisation

The CFSE-staining can withstand cryosectioning procedures

CFSE-stained keratinocytes and melanocytes could be traced in tissue

sections from in vitro wounds to up to three weeks

63

The proliferation measurement with the aid of flow cytometry of CFSE-staining is

based on the dilution of the staining that occurs at every cell division. It is assumed

that the fluorescence intensity is halved at every generation. CFSE fulfils several

important requirements for a dye to perform with high resolution under these

premises: the initial staining intensity is high, the variability of the fluorescence is

low, there is minimal transfer of dye after staining and CFSE shows low toxicity

for cells (254). Due to staining intensity and low variability in fluorescence,

distinct peaks in fluorescence intensity can be obtained when investigating number

of cell divisions with flow cytometry. In paper II we have investigated the presence

of stained cells with retained fluorescent signal in tissue sections, unable to state

how many cell divisions the observed cell has undergone. Measurements of

staining intensity of traced cells in sections needs to be compared with newly

stained cells, to be able to report number of divisions of transplanted cells that have

taken place. The performance of CFSE as a staining for measuring fluorescence

intensity in tissue sections needs to be evaluated. The application of CFSE-staining

on human primary epidermal cells could be a valuable tool for investigating

transplantation and the fate of transplanted cells owing to its simple processing,

stable fluorescence and low impact on human primary keratinocytes and

melanocytes.

Paper III: Influence of acidic pH on keratinocyte function and re-

epithelialisation of in vitro wounds

Adjuvant therapies for modifying the wound healing environment and therefore

hopefully aiding chronic wound healing include laser and ultrasound therapy,

electrical stimulation, and treatment with hyperbaric oxygen, negative pressure

therapy (255) and pH modulation (256). None of the treatment approaches have

been shown to conclusively contribute to improved healing though negative

pressure therapy has been extensively used in the clinical setting (257). Few

64

investigative reports are available on the impact on lowering wound pH on wound

healing (258). The rationale behind the lowering of pH in the wound environment

stems from the facts that intact skin is slightly acidic and that when wound healing

proceeds, a slight acidosis takes place. Several proteases and bacteria are active in

basic milieus, and chronic wounds display elevated pH levels that could contribute

to the unfavourable wound healing environment present in chronic wounds (256).

The aim of paper III was to utilise the in vitro wound healing model to investigate

re-epithelialisation of in vitro wounds exposed to pH 5.0 and pH 6.0, and the

effects on primary human keratinocytes in culture exposed to the lowered pH

levels with functional assays and qRT-PCR.

In vitro wounds were cultured submerged in FM with 10 % FCS adjusted to pH

5.0 and pH 6.0 with 1 M hydrochloric acid. Wounds cultured at pH 5.0 showed no

re-epithelialisation at any time point. Wounds and biopsy edges of wounds

cultured at pH 6.0 showed moderate outgrowth compared with control wounds

cultured at pH 7.4 (Figure 16 A, B and D). The findings in the functional assays

were supportive of the tissue culture results where the responses of keratinocytes

cultured at pH 5.0 to activation and migration cues were lost. These responses were

partially retained in keratinocytes cultured at pH 6.0 represented by moderate re-

epithelialisation of in vitro wounds.

Scratch assays revealed keratinocytes exposed to pH 6.0 to be able to repopulate

the scratched area up to 80 % after 24 hours compared with no migration taking

place in the cultures exposed to pH 5.0 (Figure 17). Reflected in the viability

measurements where viability in the pH 5.0 culture was beneath 50 % of the

normal controls at all time points, the poor migration was expected.

The qRT-PCR revealed differential expression of the investigated mRNA. The

detrimental effect of pH 5.0 and the low RNA yields it resulted in, limited the time

of exposure to lowered pH to six hours. Genes of interest were matrix

65

metalloproteinase 1 (MMP1), tissue inhibitor of matrix metalloproteinase 1

(TIMP1) (259), protein kinase 2 (PTK2) and heat shock 27 kDa protein 1 (HSPB1).

HSPB1 is also known as HSP27, heat shock protein 27, and is a cytoprotective

protein that localises to the nucleus when cells are stress induced and is involved

in avoiding apoptosis, and actin network rearrangement (260). QRT-PCR showed

upregulation of HSPB1 in cells exposed to pH 6.0. This could partly account for

the retained capacity to migrate by the cells exposed to pH 6.0 which was seen in

the scratch assay, coupled to the upregulation of PTK2 that was also revealed by

the qRT-PCR. PTK2 (also known as FAK, focal adhesion kinase) binds the

activated cytoplasmic tail of plasma membrane integrins (261) and is the mediator

in several important pathways (262) wherein migration and adhesion in response

to cues from the ECM through integrins are well charted (263). Capacity to migrate

Figure 16. Haematoxylin and eosin staining of in vitro wound sections. (A)

Neoepidermal outgrowth of wound cultured at pH 6.0 for eight days and (B)

control wound at day four. (C) Wound edges of wound exposed to pH 5.0 with no

outgrowth compared with (D) fully re-epithelialised control wound at day 12.

Arrows indicate wound edges, arrow heads neoepidermal outgrowth. Scale bar =

500 µm.

66

at pH 6.0 is of importance if acidification

is to be applied as a treatment strategy.

However, values of the qRT-PCR present

substantial variance but the results indicate

upregulation of both PTK2 and HSPB1.

Keratinocytes cultured at pH 6.0 showed

higher viability, an 80 % coverage of

scratched area in the scratch assay,

upregulation of HSPB1 and less aberrant

expression of the junctional protein ZO-1

than keratinocytes cultured at pH 5.0, with

a pattern of loss of response in cells at pH

5.0 and a recovery and adaptation of cells

to the environment at pH 6.0. Considering

the demanding environment pH 5.0

imposes the findings of poor cell function

at pH 5.0 were expected, however the

recovery of migration at pH 6.0 shows that

keratinocytes under these conditions are

responsive to cues from the environment to

migrate.

Conclusions and future outlook

No re-epithelialisation of in vitro wounds occurred under exposure to pH

5.0 compared to a minor outgrowth of an epithelial tongue in wounds

cultured at pH 6.0

Keratinocytes exposed to pH 6.0 upregulated the cytoprotective protein

HSPB1 and PTK2, important for migration

Figure 17. Scratch assay performed on keratinocytes exposed to pH 6.0 and pH 5.0. At 18 hours keratinocytes cultured at pH 6.0 repopulated the scratched area to 80 %, no migration could be observed in cultures cultured at pH 5.0.

67

Keratinocytes exposed to pH 6.0 in culture showed retained capacity to

migrate compared with keratinocytes exposed to pH 5.0

Lowering the pH levels in the chronic wound is a relevant strategy to inhibit

detrimental proteases active at high pH levels (264). In attempt to mimic the

natural state of the wound, acidification could be beneficial for chronic wound

environments that have stagnated in the inflammatory phase. According to our

findings on keratinocyte function and re-epithelialisation of in vitro wounds

exposed to pH 5.0 and 6.0 we conclude that lowering the pH in the wound

environment below pH 6.0 is inadvisable due to the severely impaired function of

keratinocytes at pH 5.0. The wound environment pH levels should be taken into

account when choosing dressings for treatment of chronic wounds in regard to

permeability of the dressing. Non-permeable dressings retain gases in the wound,

including CO2. The CO2 will contribute to acidification of the wound whereas

permeable dressings like hydrogel dressings permit gas exchange and can

contribute to maintaining pH levels in the wound healing environment suitable for

re-epithelialisation.

Paper IV: Non-occlusive topical exposure of human skin in vitro

as model for cytotoxicity testing of irritant compounds

In paper IV, human full thickness skin was utilised to investigate cytotoxicity in

skin when exposed to a known irritant. The aim was to elucidate whether intact

skin with a fully functional barrier could be used to model the response. Irritants

are difficult to test: the use of animals should be restricted from an ethical stand

point, the Draize test has been challenged over the years. Moreover, the signs of

irritation visible to the subjective investigator include erythema, oedema and

spongiosis that are difficult to discern (265). This limits the use of human subjects

where only a certain degree of discomfort is acceptable.

68

The first 15 minute exposure tests, which is standard testing time for short

exposure, on intact human skin in vitro were carried out with PBS as negative

control substance and SDS, the known irritant, diluted in PBS. The variance in

response to PBS was found to be substantial and therefore PBS as control

substance was changed to non-supplemented DMEM. The viability values

obtained from the negative controls with DMEM were more stable, and long term

(six hour) exposure experiments were carried out with DMEM as control and

solvent. Non-linear regression analysis revealed an R2 = 0.76 between increased

concentration of SDS and decreased viability of keratinocytes in the exposed area.

The exposure of intact skin to 2 % SDS only elicited a modest cytotoxic response

(Figure 18 A and B). This points out the problems that arise when skin equivalents

are used for cytotoxicity testing: the lack of full barrier protection of the skin

equivalent models carries an over-prediction when testing irritants. There are

several reconstituted human skin models, out of which EPISKIN (Episkin SNC,

Chaponost, France) and EpiDerm (MatTek Corporation, Ashland, MA) are the

most used (266). In early validation studies of the models the over-prediction rates

were between 47 % and 60 %, even if accuracy rates were acceptable (267). The

reconstituted skin models are based on normal human keratinocytes cultured on a

collagen matrix and stratified by culturing in the air-liquid interface. The epidermal

component of both systems is histologically similar to native epidermis but the

models lack in barrier structure and a complete dermal component. The barrier

capacity fails to reach the level of that of native tissue, and additionally all

interaction between keratinocytes and fibroblasts. Native target tissue is preferable

and there is a real possibility to use it in the case of human skin. In the presented

model, tissue is viable and includes all cells present in the skin except homed

immune cells (268). Most importantly the barrier components of stratum corneum,

tight junction seals, an intact basement membrane and dermal cells are all present.

69

The foremost objection to using human skin in culture for testing of irritant

potential is the inherent interpersonal variability. To counteract this, every

experiment was performed on one skin sample. The irritant response measured by

cytotoxicity was related to an internal positive control at all times. The positive

control was 20 % SDS in DMEM. SDS is one of the most utilised known irritants

in irritant testing, and 20 % is a frequently used concentration to elicit an irritant

reaction (269). The reaction was then related to a negative control to obtain

information on irritant potential, and also relate the investigated

compound/concentration to the positive control to ensure the level of irritation the

investigated compound evokes. In the application of human full thickness skin for

Figure 18. (A) Distribution of viability values of all six hour exposure experiments with full thickness skin exposed to 2 %, 10 % and 20 % sodium dodecyl sulphate (SDS). Viability values are relative to control Dulbecco’s modified Eagle medium

(DMEM). All group means differ significantly from control DMEM (mean ± SD, n = 72). (B) Non-linear regression analysis of increased concentration of SDS and decreased viability, R2 = 0.76.

70

irritant testing cytotoxicity was chosen as parameter to investigate. It is a

quantifiable parameter that directly reflects the damage to the keratinocytes.

Disturbance of keratinocytes also initiates the subsequent events that lead to the

manifestation of irritation by causing release of stored IL-1α from the

keratinocytes. A limitation of using human skin in vitro, or any other in vitro

model, is the lack of circulation and a systemic response. However, a limited

immune response in human skin in vitro is present in the form of stochastic

presence of immune cells. Compared to reconstituted skin models this is still an

advantage that the full thickness skin presents. By limiting the exposure to only

topical the entry route of the investigated substance is the same that would occur

in a real life situation and the immune activation starts with the release of cytokines

from disrupted keratinocytes. The interaction of keratinocytes with melanocytes

and dermal fibroblasts can take place in a model of full thickness skin, and the

release of cytokines by keratinocytes and the subsequent effects on fibroblasts is

of interest to develop the human full thickness irritation model further.

Conclusions and future outlook

Viability staining of skin samples topically exposed to irritant yielded

stable responses with non-supplemented Dulbecco’s modified Eagle

medium as negative control substance

Non-linear regression analysis revealed R2 = 0.76 between increased

concentration of known irritant sodium dodecyl sulphate and decreased

cell viability in the exposed areas

Many different approaches for toxicity testing are being developed to replace the

use of animals. The next step in any development of a new methodology is the

validation of the model. This includes systematic testing with known reference

chemicals to determine the accuracy of the model and the intra- and inter

71

laboratory variability. The validation process should also establish the suitability

of the model to test different classes of chemicals. The use of viable human skin

as presented in paper IV needs to be validated to adhere to regulatory settings.

In the present study the means of exposure of the human full thickness skin was

non-occluded, a mode of application that cannot easily be performed on human

subjects. The presented model can be modified to investigate occluded exposure

as well as repeated exposures. Repeated and occluded exposures are both relevant

problem formulations for occupational hazard investigations. Human full

thickness skin can be kept in culture for several weeks which enables the model to

be utilised for long term exposures, another exposure scheme that is problematic

to perform on human volunteers. Additionally, human skin in vitro can be pre-

disposed, with e.g. water exposure, or abraded to investigate more specific

question formulations where the quality of skin plays a part. The proposed model

can be easily modified to provide more insight in irritant cytotoxicity in human

skin.

72

73

CONCLUDING REMARKS

The work presented within this thesis has focused on applications of human full

thickness skin in vitro. Human skin in vitro presents an adequate response to

wounding visualised by the re-epithelialisation of standardised wounds and

possesses a fully functional cutaneous barrier. Representative model systems for

any physiological or pathological process is a necessity for relevant research focus

and translational success. In the case of cutaneous wound healing, the need for a

modelling system that takes into account the reciprocal interactions that take place

during the complex process is crucial, as re-epithelialisation is highly dependent

on extracellular matrix cues and fibroblast interaction. Investigations on wound

healing and irritant response employing human skin in vitro makes use of the

actual target tissue with all constituting cell types and matrix components present.

74

ACKNOWLEDGEMENTS

I would like to extend a sincere thank you to the following persons who have

contributed to this thesis and supported my work:

My main supervisor Gunnar Kratz, for giving me the opportunity to work with

you and the freedom to develop, for the encouragement and all the lessons on

positive thinking.

My co-supervisor Magnus Berggren, for introducing me to the world of organic

electronics and the people working in your group, your professionalism is truly

inspiring.

Jonathan Rakar my colleague, supervisor, collaborator and friend, for all work

you have done and all support (both IT and mental) you have provided over these

years.

Kristina Briheim for teaching me all I know and more about cell and tissue

culture, and for being a stand in parent.

All members of the plastic surgery lab I have had the pleasure to meet and work

with over the years, Johan Junker for supervision, introduction to coffee induced

science, and proof reading, Anita Lönn for all assistance and introduction to

teaching, Pehr Sommar and Lisa Karlsson for collaborations, Maria Karlsson for

contribution to paper II.

Kristin Persson, David Nilsson, Anurak Sawatdee, Henrik Toss, Josefin Nissa

& Daniel Simon at the Laboratory of Organic Electronics, for interesting and

challenging collaborations.

Stefan Klintström, Charlotte Immerstrand & Anette Svensson of Forum

Scientium for inspirational study visits.

Everyone at KEF for creating a nice place to work, Stina & Tove who paved the

way, Sebastian & Cynthia for the good times, after works and proof reading.

Micke Pihl at the Flow facility for first suggesting CFSE and help with flow

cytometry. Håkan Wiktander and Åsa Schippert & Anette Molbaek at Core

facility for help with really everything.

75

Linda, Pilvi, Vivi & Linda, Anna, Katarina for being part of the home to come

home to.

Jonas, Anders, Jocke & Sandra, Joanne & Ryan my beloved dysfunctional

family!

Sofie, Linnea & Cici for excellent peer support and invaluable friendship.

Äiti, Linda & Pappa for never questioning and always supporting.

Johannes there is no one whose opinion or advice I value higher, it is all for you!

76

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Papers

The articles associated with this thesis have been removed for copyright reasons. For more details about these see: http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-123313